Polycystic kidney disease 1 gene and uses thereof

ABSTRACT

The present invention relates to the polycystic kidney disease 1 (PKD1) gene and its nucleic acid sequence, mutations thereof in patients having PKD1-associated disorders, the protein encoded by the PKD1 gene or its mutants, and their uses in disease diagnosis and therapy.

This application is a CIP of international application number PCT GB94/02822, filed Dec. 23, 1994.

BACKGROUND TO THE INVENTION

In humans, one of the commonest of all genetic disorders is autosomal polycystic kidney disease (ADPKD) also termed adult polycystic kidney disease (APKD), affecting approximately 1/1000 individuals (Dalgaard, 1957). ADPKD is a progressive disease of cyst formation and enlargement typically leading to end stage renal disease (ESRD) in late middle age. The major cause of morbidity in ADPKD is progressive renal disease characterized by the formation and enlargement of fluid filled cysts, resulting in grossly enlarged kidneys. Renal function deteriorates as normal tissue is compromised by cystic growth, resulting in end stage renal disease (ESRD) in more than 50% of patients by the age of 60 years (Gabow, et al., 1992). ADPKD accounts for 8-10% of all renal transplantation and dialysis patients in Europe and the USA (Gabow, 1993).

ADPKD also causes cystic growth in other organs (reviewed in Gabow, 1990) and occasionally presents in childhood (Fink, et al., 1993; Zerres, et al., 1993). Extrarenal manifestations include liver cysts (Milutinovic, et al., 1980), and more rarely cysts of the pancreas (Gabow, 1993) and other organs. Intracranial aneurysms occur in approximately 5% of patients and are a significant cause of morbidity and mortality due to subarachnoid haemorrhage (Chapman, et al., 1992). ADPKD is associated with a higher prevalence of various connective tissue disorders. An increased prevalence of heart valve defects (Hossack, et al., 1988), hernia (Gabow, 1990) and colonic diverticulae (Scheff, et al., 1980) have been reported.

Considerable progress has been made in the last few years in understanding the pathophysiology of ADPKD (and other animal models of cystic disease). Cysts in ADPKD are known to develop from outpouchings of descending or ascending kidney tubules and the early stages are characterized by a thickening and disorganization of the basement membrane, accompanied by a de-differentiation of tubular epithelial cells. Several of the characteristics of ADPKD epithelia: altered growth responses, abnormal expression of various proteins and reversal of polarity, may be a sign of this de-differentiation and important in cyst expansion. The nature of the primary defect which triggers these changes is, however, unknown and consequently much effort has been devoted to identifying the causative agent by genetic means.

The first step towards positional cloning of an ADPKD gene was the demonstration of linkage of one locus now designated the polycystic kidney disease 1 (PKD1) locus to the α globin cluster on the short arm of chromosome 16 (Reeders, et al., 1985). Subsequently, families with ADPKD unlinked to markers one of 16p were described (Kimberling, et al., 1988; Romeo, et al., 1988) and a second ADPKD locus (PKD2) has recently been assigned to chromosome region 4q13-q23 (Kimberling, et al., 1993; Peter, et al., 1993). It is estimated that approximately 85% of ADPKD is due to PKD1 (Peters and Sankuijl, 1992) with PKD2 accounting for most of the remainder. PKD2 appears to be milder condition with a later age of onset and ESRD (Parfrey, et al., 1990; Gabow, et al., 1992; Ravine, et al., 1992).

The position of the PKD1 locus was refined to chromosome band 16p13.3 and many markers were isolated from that region (Breuning, et al., 1987; Reeders, et al., 1988; Breuning, et al., 1990; Germino, et al., 1990; Hyland, et al., 1990; Himmelbauer, et al., 1991). Their order, and the position of the PKD1 locus, has been determined by extensive linkage analysis in normal and PKD1 families and by the use of a panel of somatic cell hybrids (Reeders et al., 1988; Breuning, et al., 1990; Germino, et al., 1990). ADPKD is genetically heterogenous with loci mapped not only to 16p13.3 (PKD1), but also to chromosome 4 (PKD2). Although the phenotype of PKD1 and PKD2 are clearly similar, it is now well documented that PKD1 (which accounts for about 85% of ADPKD; (Peters, 1992) is a more severe disease with an average age at ESRD of about 56 years compared to about 71.5 years for PKD2 (Ravine, 1992). An accurate long range restriction map of the 16p13.3 region (Harris, et al., 1990; Germino, et al., 1992) has located the PKD1 locus in an interval of approximately 600 kb between the markers GGG1 and SM7 (Harris, et al., 1991; Somlo, et al., 1992) (see FIG. 1a). The density of CpG islands and identification of many mRNA transcripts indicated that this area is rich in gene sequences. Germino et al. (1992) estimated that the candidate region contains approximately 20 genes.

Identification of the PKD1 gene from within this area has thus proved difficult and other means to pinpoint the disease gene have been sought. Linkage disequilibrium has been demonstrated between PKD1 and the proximal marker VK5, in a Scottish population (Pound, et al., 1992) and between PKD1 and BLu24 (see FIG. 1a), in a Spanish population (Peral, et al., 1994). Studies with additional markers have shown evidence of a common ancestor in a proportion of each population (Peral, et al., 1994; Snarey, et al., 1994), but the association has not precisely positioned the PKD1 locus.

Disease associated genomic rearrangements, detected by cytogenetics or pulsed field gel electrophoresis (PFGE) have been instrumental in the identification of various genes associated with various genetic disorders. Hitherto, no such abnormalities related to PKD1 have been described. This situation contrasts with that for the tuberous sclerosis locus, which lies within 16p13.3 (TSC2). In that case, TSC associated deletions were detected by PFGE within the interval thought to contain the PKD1 gene and their characterisation was a significant step toward the rapid identification of the TSC2 gene (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). The TSC2 gene therefore maps within the candidate region for the hitherto unidentified PKD1 gene; as polycystic kidneys are a feature common to TSC and ADPKD1 (Bernstein and Robbins, 1991) the possibility of an etiological link, as proposed by Kandt et al. (1992), was considered. A contiguous gene syndrome resulting from the disruption of PKD1 and the adjacent tuberous sclerosis 2 (TSC2) gene, which is associated with TSC and severe childhood onset polycystic kidney disease, has also been defined (Brook-Carter et al, 1994).

We have now identified a pedigree in which the two distinct phenotypes, typical ADPKD or TSC, are seen in different members. In this family, the two individuals with ADPKD are carriers of a balanced chromosome translocation with a breakpoint within 16p13.3. We have located the chromosome 16 translocation breakpoint and a gene disrupted by this rearrangement has been defined; the discovery of additional mutations of that gene in other PKD1 patients shows that we have identified the PKD1 gene. Full characterisation of the PKD1 transcript has been significantly complicated because of the unusual genomic region containing most of the gene. All but 3.5 kb at the 3′ end of the transcript (which is about 14 kb in total) is encoded by a region which is reiterated several times elsewhere on the same chromosome (in 16p13.1 and termed the HG area). The structure of the duplication is complex, with some regions copied more times than others, and the HG region encoding three large transcripts. The transcripts from the HG area are: HG-A (21 kb), HG-B (17 kb) and HG-C (8.5 kb) and although these have 3′ ends which differ from PKD1, over most of their length they share substantial homology to the PKD1 transcript. Consequently, cloning and characterizing a bona fide PKD1 cDNA has proven difficult. To overcome the problem caused by duplication we have cloned cDNAs covering the entire transcript from a cell line which contains the PKD1 but not the HG loci. Characterisation of these cDNAs has enabled the PKD1 protein sequence to be predicted and led to the identification of several homologies with described motifs.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, this invention provides an isolated, purified or recombinant nucleic acid sequence comprising:

(a) a PKD1-encoding nucleic acid or its complementary strand,

(b) a sequence substantially homologous to, or capable of hybridizing to, a substantial portion of a molecule defined in (a) above, or

(c) a fragment of a molecule defined in (a) or (b) above.

In particular, there is provided a sequence wherein the PKD1 gene has the nucleic acid sequence according to FIG. 15 (SEQ ID NO. 7), or the partial sequence of FIGS. 7 (SEQ ID NO. 1) or 10 (SEQ ID NO. 5). The invention therefore includes a DNA molecule coding for a polypeptide having the amino acid sequence of FIG. 15 (SEQ ID NO. 8), or a polypeptide fragment thereof; and genomic DNA corresponding to a molecule as in (a)-(c) above.

As used herein, “substantially homologous” refers to a nucleic acid strand that is sufficiently duplicative of the PKD1 sequence presented in FIG. 15 (SEQ ID NO. 7) such that it is capable of hybridizing to that sequence under moderately stringent, and preferably stringent conditions, as defined herein below. Preferably “substantially homologous” refers to a homology of between 97 and 100%. Further, such a strand will encode or be complementary to a strand that encodes PKD1 protein having the biological activity described below. As used herein, a “substantial portion of a molecule” refers to at least 60%, preferably 80% and most preferably 90% of the molecule in terms of its linear residue length or its molecular weight. “Nucleic acid” refers to both DNA and RNA.

The PKD1 gene described herein is a gene found on human chromosome 16, and the results of studies described herein form the basis for concluding that this PKD1 gene encodes a protein called PKD1 protein which has a role in the prevention or suppression of ADPKD. The PKD1 gene therefore includes the DNA sequences shown in FIG. 15 (SEQ ID NO. 7), and all functional equivalents. By “functional equivalents”, we mean nucleic acid sequences that are substantially homologous to the PKD1 nucleic acid sequence, as presented in FIG. 15 (SEQ ID NO. 7), and encoding a protein that possesses one or more of the biological functions or activities of PKD1; i.e., that is involved in cell/cell adhesion, cell/cell recognition or cell/cell communication, for example to effect adhesion of cells to other cells or components of the extracellular matrix; effect communication and/or interaction between epithelial cells and the basal membrane (whether in kidneys or otherwise); assist in development of connective tissue such as assembly and/or maintenance of the basal membrane; in signal transduction between cells or cells and components of the extracellular matrix; and/or to promote binding of cells carrying proteins such as integrins or carbohydrates to target cells. The biological function of PKD1 of course includes maintaining a healthy physiological state; that is, the native protein's aberrations or absence results in ADPKD or an associated disorder.

The PKD1 gene may furthermore include regulatory regions which control the expression of the PKD1 coding sequence, including promoter, enhancer and terminator regions. Other DNA sequences such as introns spliced from the end-product PKD1 RNA transcript are also encompassed. Although work has been carried out in relation to the human gene, the corresponding genetic and functional sequences present in lower animals are also encompassed.

The present invention therefore further provides a PKD1 gene or its complementary strand having the sequence according to FIG. 15 (SEQ ID NO. 7) which gene or strand is mutated in some ADPKD patients (more specifically, PKD1 patients). Therefore, the invention further provides a nucleic acid sequence comprising a mutant PKD1 gene as described herein, including wherein Intron 43 as defined hereinbelow has a deletion of 18 or 20 bp resulting in an intron of 55 or 57 bp.

As used herein, “PKD1 mutant” or “mutation” encompasses alterations of the native PKD1 nucleotide (SEQ ID NO. 7) or amino acid sequence (SEQ ID NO. 8), as defined by FIG. 15, i.e., substitutions, deletions or additions, and also encompasses deletion of DNA containing the entire PKD1 gene.

The invention further provides a nucleic acid sequence comprising a mutant PKD1 gene, especially one selected from a sequence comprising a partial sequence according to FIGS. 7 (SEQ ID NO. 1) and/or 10 (SEQ ID NO. 5), or the corresponding sequences disclosed in FIG. 15 (SEQ ID NO. 7), when:

(a) [OX114] base pairs 1746-2192 as defined in FIG. 7 (SEQ ID NO. 1) are deleted (446 bp);

(b) [OX32] base pairs 3696-3831 as defined in FIG. 7 (SEQ ID NO. 1) are deleted by a splicing defect;

(c) [OX875] about 5.5 kb flanked by the two Xbal sites shown in FIG. 3a are deleted and the EcoR1 site separating the CW10 (41 kb) and JH1 (18 kb) sites is thereby absent

(d) [WS53] about 100 kb extending between the JH1 and CW21 and the SM6 and JH17 sites shown in FIG. 6 and the PKD1 gene is thereby absent, the deletion lying proximally between SM6 and JH17;

(e) [461] 18 bp are deleted in the 75 bp intron amplified by the primer pair 3A3C (SEQ ID NOS. 11 and 12) insert at position 3696 of the 3′ sequence (SEQ ID NO. 1) as shown in FIG. 11;

(f) [OX1054] 20 bp are deleted in the 75 bp intron amplified by the primer pair 3A3C insert at position 3696 of the 3′ sequence as shown in FIG. 11 (SEQ ID NO. 18);

(g) [WS212] about 75 kb are deleted between SM9-CW9 distally and the PKD1 3′UTR proximally as shown in FIG. 12;

(h) [WS-215] about 160 kb are deleted between CW20 and SM6-JH17 as shown in FIG. 12;

(i) [WS-227] about 50 kb are deleted between CW20 and JH11 as shown in FIG. 12;

(j) [WS-219] about 27 kb are deleted between JH1 and JH6 as shown in FIG. 12;

(k) [WS-250] about 160 kb are deleted between CW20 and Blu24 as shown in FIG. 12;

(l) [WS-194] about 65 kb is deleted between CW20 and CW10.

The invention therefore extends to RNA molecules comprising an RNA sequence corresponding to any of the DNA sequences set out above. Such molecule may be the transcript reference PBP and identifiable with respect to the restriction map of FIG. 3a and having a length of about 14 kb.

In another aspect, the invention provides a nucleic acid probe having a sequence as set out above; in particular, this invention extends to a purified nucleic acid probe which hybridizes to at least a portion of the DNA or RNA molecule of any of the preceding sequences. Preferably, the probe includes a label such as a radiolabel, for example, a ³²P label.

In another aspect, this invention provides a purified DNA or RNA coding for a protein comprising the amino acid sequence of FIG. 15 (SEQ ID NO. 8), or a protein polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein.

The DNA molecule defined above may be incorporated in a recombinant cloning vector for expressing a protein having the amino acid sequence of FIG. 15 (SEQ ID NO. 8), or a protein or a polypeptide having at least one functional domain or active site in common with said protein. Such a vector may include any vector for expression in bacteria, e.g., E. coli; yeast, insect, or mammalian cells.

The invention also features a nucleic acid probe for detecting PKD1 nucleic acid comprising 10 consecutive nucleotides as presented in FIG. 15 (SEQ ID NO. 7). Preferably, the probe may comprise 15, 20, 50, 100, 200, or 300, etc., consecutive nucleotides (nt) presented in FIG. 13, and may fall within the size range 15 nt-13 kb, 100 nt-5 kb, 150 nt-4 kb, 300 nt-2 kb, and 500 nt-1 kb.

Probes are used according to the invention in hybridization reactions to identify PKD1 sequences, whether they be native or mutated PKD1 DNA or RNA, as disclosed herein. Such probes are useful for identifying the PKD1 gene or a mutation thereof, as defined herein.

The invention also features a synthetic polypeptide corresponding in amino acid residue sequence to at least a portion of the sequence of naturally occurring PKD1, and having a molecular weight equal to less than that of the native protein. A synthetic polypeptide of the invention is useful for inducing the production of antibodies specific for the synthetic polypeptide and that bind to naturally occurring PKD1.

Preferred embodiments of this aspect of the invention include a group of synthetic polypeptides whose members correspond to a fragment of the PKD1 protein comprising a stretch of amino acids of at least 8, and preferably 15, 30, 50, or 100 residues in length from the sequence disclosed in FIG. 15 (SEQ ID NO. 8).

In another aspect, the invention provides a polypeptide encoded by a sequence as set out above, or having the amino acid sequence according to the amino acid sequence of FIG. 15 (SEQ ID NO. 8), or a protein or polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein. In particular, there is provided an isolated, purified or recombinant polypeptide comprising a PKD1 protein or a mutant or variant thereof or encoded by a sequence set out above or a variant thereof having substantially the same activity as the PKDL protein. The present invention may further comprise a polypeptide having 9 or 13 transmembrane pairs instead of 11 transmembrane domains as described hereinbelow. Further comprising this invention is a molecule which interacts with a polypeptide as herein described which molecule synergises, causes, enhances or is necessary for the functioning of the PKD1 protein as herein described.

The invention also encompasses recombinant expression vectors comprising a nucleic acid or isolated DNA encoding PKD1 and a process for preparing PKD1 polypeptide, comprising culturing a suitable host cell comprising the vector under conditions suitable for promoting expression of PKD1, and recovering said PKD1.

This invention also provides an in vitro method of determining whether an individual is likely to be affected with tuberous sclerosis, comprising assaying a biological sample from the individual to determine the presence and/or amount of PKD1 protein or polypeptide having the amino acid sequence of FIG. 15 (SEQ ID NO. 8).

As used herein, “biological sample” includes any fluid or tissue sample from a mammal, preferably a human, including but not limited to blood, urine, saliva, any body organ tissue, cells from any body tissue, including blood cells.

Additionally or alternatively, a sample may be assayed to determine the presence and/or amount of mRNA coding for the protein or polypeptide having the amino acid sequence of FIG. 15 (SEQ ID NO. 8), or to determine the fragment lengths of fragments of nucleotide sequences coding for the protein or polypeptide of FIG. 15 (SEQ ID NO. 8), or to detect inactivating mutations in DNA coding for a protein having the amino acid sequence of FIG. 15 (SEQ ID NO. 8) or a protein having homologous properties. The screening preferably includes applying a nucleic acid amplification process, as described herein in detail, to said sample to amplify a fragment of the DNA sequence. The nucleic acid amplification process advantageously utilizes at least one of the following sets of primers as identified herein: AH3 F9 (SEQ ID NO. 9):AH3 B7 (SEQ ID NO. 10); 3A3 C1 (Seq ID No: 11):3A3 C2 (Seq ID No: 12); and AH4 F2 (Seq ID No:13):JH14 B3 (Seq ID No: 14).

Alternatively, the screening method may comprise digesting the sample DNA to provide EcoRI fragments and hybridizing with a DNA probe which hybridizes to the EcoRI fragment identified (A) in FIG. 3(a), and the DNA probe may comprise the DNA probe CW10 (SEQ ID NO. 4) identified herein.

Another screening method may comprise digesting the sample to provide BamHI fragments and hybridizing with a DNA probe which hybridizes to the BamHI fragment identified (B) in FIG. 3(a), and the DNA probe may comprise the DNA probe 1A1H.6 identified herein.

A method according to the present invention may comprise detecting a PKD1-associated disorder in a patient suspected of having or having predisposition to the disorder (i.e., a carrier), the method comprising detecting the presence of and/or evaluating the characteristics of PKD1 DNA, PKD1 mRNA and.or PKD1 protein in a sample taken from the patient. Such method may comprise detecting and/or evaluating whether the PKD1 DNA is deleted, missing, mutated, aberrant or not expressing normal PKD1 protein. One way of carrying out such a method comprises: A. taking a biological, tissue or biopsy sample from the patient; B. detecting the presence of and/or evaluating the characteristics of PKD1 DNA, PKD1 mRNA and/or PKD1 protein in the sample to obtain a first set of results; C. comparing the first set of results with a second set of results obtained using the same or similar methodology for an individual that is not suspected of having the disorder; and if the first and second sets of results differ in that the PKD1 DNA is deleted, missing, aberrant, mutated or not expressing PKD1 protein then that is indicative of the presence, predisposition or tendency of the patient to develop the disorder. As used herein, a “PKD1-associated disorder” refers to adult polycystic kidney disease, as described herein, and also refers to tuberous sclerosis, as well as other disorders having symptoms such as cyst formation in common with these diseases.

A specific method according to the invention comprises extracting from a patient a sample of PKD1 DNA or DNA from the PKD1 locus purporting to be PKD1 DNA, cultivating the sample in vitro and analyzing the resulting protein, and comparing the resulting protein with normal PKD1 protein according to the well-established Protein Truncation Test. Less sensitive tests include analysis of RNA using RT PCR (reverse transcriptase polymerase chain reaction), and examination of genomic DNA.

Step C of the above method may be replaced by: comparing the first set of results with a second set of results obtained using the same or similar methodology in an individual that is known to have the or at least one of the disorder(s); and if the first and second sets of results are substantially identical, this indicates that the PKD1 DNA in the patient is deleted, mutated or not expressing normal PKD1 protein.

The invention further provides a method of characterizing a mutation in a subject suspected of having a mutation in the PKD1 gene, which method comprises: A. amplifying each of the exons in the PKD1 gene of the subject; B. denaturing the complementary strands of the amplified exons; C. diluting the denatured separate, complementary strands to allow each single-stranded DNA molecule to assume a secondary structural confirmation; D. subjecting the DNA molecule to electrophoresis under non-denaturing conditions; E. comparing the electrophoresis pattern of the single-stranded molecule with the electrophoresis pattern of a single-stranded molecule containing the same amplified exon from a control individual which has either a normal or PKD1 heterozygous genotype; and, F. sequencing any amplification product which has an electrophoretic pattern different from the pattern obtained from the DNA of the control individual.

The invention also extends to a diagnostic kit for carrying out a method as set out above, comprising nucleic acid primers for amplifying a fragment of the DNA or RNA sequences defined above, and packaging means therefore. The kit may optionally include written instructions stating that the primers are to be used for detection of disorders associated with the PKD1 gene. The nucleic acid primers may comprise at least one of the following sets: AH3 F9 (Seq ID No:9):AH3 B7 (Seq ID No:10); 3A3 C1 (Seq ID No:11):3A3 C2 (Seq ID No:12); and AH4 F2 (Seq ID No:13):JH14 B3 (Seq ID No: 14).

Another embodiment of kit may combine one or more substances for digesting a sample to provide EcoRI fragments and a DNA probe as previously defined. A further embodiment of kit may comprise one or more substances for digesting a sample to provide BamHI fragments and a DNA probe as previously defined.

A vector (such as Bluescript (available from Stratagene)) comprising a nucleic acid sequence set out above; and a host cell (such as E. coli strain SL-1 Blue (available from Stratagene)) transfected or transformed with the vector are also provided, together with the use of such a vector or a nucleic acid sequence set out above in gene therapy and/or in the preparation of an agent for treating or preventing a PKD1-associated disorder.

Therefore, there is further provided a method of treating or preventing a PKD1-associated disorder which method comprises administering to a patient in need thereof a functional PKD1 gene to affected cells in a manner that permits expression of PKD1 protein therein and/or a transcript produced from a mutated chromosome (such as the deleted WS-212 chromosome) which is capable of expressing functional-PKD1 protein therein.

As used herein, the term “hybridization” refers to conventional DNA/DNA or DNA/RNA hybridization conditions. For example, for a DNA or RNA probe of about 10-50 nucleotides, moderately stringent hybridization conditions are preferred and include 10×SSC, 5×Denhardts, 0.1% SDS, at 35-50 degrees for 15 hours; for a probe of about 50-300 nucleotides, “stringent” hybridization conditions are preferred and refer to hybridization in 6×SSC, 5×Denhardts, 0.1% SDS at 65 degrees for 15 hours.

The present invention further provides the use of PKD1 protein or polycystin or a mutant or variant thereof having substantially the same biological activity there as in therapy. In particular, to effect cell adhesion, recognition or communication for example to effect adhesion of cells to other cells or components of the extracellular matrix; effect communication and/or interaction between epithelial cells and the basal membrane (whether in kidneys or otherwise); assisting in development of connective tissue such as assembly and/or maintenance of the basal membrane; in signal transduction between cells or cells and components of the extracellular matrix; and/or to promote binding of cells carrying proteins such as integrins or carbohydrates to target cells.

Accordingly, where it is preferred to administer the polypeptide directly to a patient in need thereof, the invention further provides the :use of a PKD1 protein or polycystin in the preparation of a medicament. Therefore, there is also provided a pharmaceutical formulation comprising a PKD1 protein, functional PKD1 gene and/or a transcript produced from a mutated chromosome which is capable of expressing functional PKD1 protein, in association with a pharmaceutically acceptable carrier therefor.

The invention also features an immunoglobin, i.e., a polyclonal or monoclonal antibody specific for an epitope of PKD1, which epitope is found in the amino acid sequence presented in FIG. 15 (SEQ ID NO. 8).

The invention also features a method of assaying for the presence of PKD1 in a sample of mammalian, preferably human cells, comprising the steps of: (a) providing an antibody specific for said PKD1; and (b) assaying for the presence of PKD1 by admixing an aliquot from a sample of mammalian cells with antibody under conditions sufficient to allow for formation and detection of an immune complex of PKD1 and the antibody. Such method is useful for detecting disorders involving aberrant expression of the PKD1 gene or processing of the protein, as described herein.

Preferably, this method includes providing a monoclonal antibody specific for an epitope that is antigenically the same, as determined by Western blot assay, ELISA or immunocytochemical staining, and substantially corresponds in amino acid sequence to the amino acid sequence of a portion of PKD1 and having a molecular weight equal to less than that of PKD1.

The invention thus also features a kit for detecting PKD1, the kit including at least one package containing an antibody or idiotype-containing polyamide portion of an antibody raised to a synthetic polypeptide of this invention or to a conjugate of that polypeptide bound to a carrier. An indicating group or label is utilized to indicate the formation of an immune reaction between the antibody and PKD1 when the antibody is admixed with tissue or cells.

Further features will become more fully apparent in the following description of the embodiments of this invention and from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Before describing preferred embodiments of the invention in detail, the drawings will briefly be described.

FIG. 1a (top): A long range map of the terminal region of the short arm of chromosome 16 showing the PKD1 candidate region defined by genetic linkage analysis. The positions of selected DNA probes and microsatellites used for haplotype, linkage or heterozygosity analyses are indicated. Markers previously described in linkage disequilibrium studies are shown in bold (from: Harris, et al., 1990; Harris, et al., 1991; Germino, et al., 1992; Somlo, et al., 1992; Peral, et al., 1994; Snarey, et al., 1994).

(bottom): A detailed map of the distal part of the PKD1 candidate region showing: the area of 16p13.3 duplicated in 16p13.1 (hatched); C, Cla I restriction sites; the breakpoints in the somatic cell hybrids, N—OH1 and P-MWH2A; DNA probes and the TSC2 gene. The limits of the position of the translocation breakpoint found in family 77 (see b), determined by evidence of heterozygosity (in 77-4) and PFGE (see c and text) is also indicated. The contig covering the 77 breakpoint region consists of the cosmids: 1, CW9D; 2, ZDS5; 3, JH2A; 4, REP59; 5, JC10.2B; 6, CW10III; 7, SM25A; 8, SMII; 9, NM17.

FIG. 1b: Pedigree of family 77 which segregates a 16;22 translocation; showing the chromosomal composition of each subject. Individuals 77-2 and 77-3 have the balanced products of the exchange—and have PKD1; 77-4 is monosomic for 16p13.3-->16pter and 22q11.21-->22pter—and has TSC.

FIG. 1c: PFGE of DNA from members of the 77 family: 77-1 (1); 77-2 (2); 77-3 (3); 77-4 (4); digested with Cla I and hybridised with SM6. In addition to the normal fragments of 340 and partially digested fragment of 480 kb a proximal breakpoint fragment of approximately 100 kb (arrowed) is seen in individuals, 77-2, 77-3 and 77-4; concordant with segregation of the der(16) chromosome.

FIG. 2: FISH of the cosmid CW10III (cosmid 6; FIG. 1a) to a normal male metaphase. Duplication of this locus is illustrated with two sites of hybridisation on 16p; the distal site (the PKD1 region) is arrowed. The signal from the proximal site (16p13.1) is stronger than that from the distal, indicating that sequences homologous to CW10III are reiterated in 16p13.1.

FIG. 3a: A detailed map of the 77 translocation region showing the precise localisation of the 77 breakpoint and the region that is duplicated in 16p13.1 (hatched). DNA probes (open boxes); the transcripts, PKD1 and TSC2 (filled boxes; with direction of transcription indicated by an arrow) and cDNAs (grey boxes) are shown below the genomic map. The known genomic extent of each gene is indicated at the bottom of the diagram and the approximate genomic locations of each cDNA is indicated under the genomic map. The positions of genomic deletions found in PKD1 patients, OX875 and OX114, are also indicated. Restriction sites for EcoR I (E) and incomplete maps for BamH I (B); Sac I (S) and Xba I (X) are shown. SM3 is a 2 kb BamH1 fragment shown at the 5′ end of the gene.

FIG. 3b: Southern blots of BamH I digested DNA from individuals: 77-1 (1); 77-2 (2); and 77-4 (4) hybridised with: left panel, 8S3 and right panel, 8S1 (see a). 8S3 detects a novel fragment on the telomeric side of the breakpoint (12 kb: arrowed) associated with the der(22) chromosome in 77-2, but not 77-4; 8S1 identifies a novel fragment on the centromeric side of the breakpoint (9 kb: arrowed)—associated with the der(16) chromosome—in 77-2 and 77-4. The telomeric breakpoint fragment is also seen weakly with 8S1 (arrowed) indicating that the breakpoint lies in the distal part of 8S1. The 8S3 and 8S1 loci are both duplicated; the normal BamH I fragment detected at the 16p13.3 site by these probes is 11 kb (see a), but a similar sized fragment is also detected at the 16p13.1 site. Consequently, the breakpoint fragments are much fainter than the normal (16p13.1 plus 16p13.3) band.

FIG. 4a: PBP cDNA, 3A3, hybridised to a Northern blot containing about 1 μg polyA selected mRNA per lane of the tissue specific cell lines: lane 1, MJ, EBV-transformed lymphocytes; lane 2, K562, erythroleukemia; lane 3, FS1, normal fibroblasts; lane 4, HeLa, cervical carcinoma; lane 5, G401, renal Wilm's tumour; lane 6, Hep3B, hepatoma; lane 7, HT29, colonic adenocarcinoma; lane 8, SW13, adrenal carcinoma; lane 9, G-CCM, astrocytoma. A single transcript of approximately 14 kb is seen; the highest level of expression is in fibroblasts and in the astrocytoma cell line, G-CCM. Although in this comparative experiment little expression is seen in lanes 1, 4 and 7, we have demonstrated at least a low level of expression in these cell lines on other Northern blots and by RT-PCR (see later).

FIG. 4b: A Northern blot containing about 20 μg of total RNA from the cell line G-CCM hybridised with cDNAs or a genomic probe which identify various parts of the PBP gene. Left panel, a single about 14 kb transcript is seen with a cDNA from the single copy area, 3A3. Right panel, a cDNA, 21P.9, that is homologous to parts of the region that is duplicated (JH12, JH8 and JH10; see FIG. 3a) hybridises to the PBP transcript and three novel transcripts; HG-A (about 21 kb), HG-B (about 17 kb) and HG-C (8.5 kb). A similar pattern of transcripts is seen with cDNAs and genomic fragments that hybridise to the area between JH5 and JH13, with the exception of the JH8 area. Middle panel, JH8 hybridises to the transcripts PBP, HG-A and HG-B but not to HG-C.

FIG. 4c: A Northern blot of 20 μg total fibroblast RNA from: normal control (N); 77-2 (2); 77-4 (4) hybridised with 8S1, which contains the 16;22 translocation breakpoint (see FIG. 3). A transcript of about 9 kb (PBP-77) is identified in the two patients with this translocation but not in the normal control. PBP-77 is a chimeric PBP transcript formed due to the translocation and is not seen in 77-2 or 77-4 RNA with probes which map distal to the breakpoint.

FIG. 5a: FIGE of DNA from: normal (N) and ADPKD patient OX875 (875), digested with EcoR I and hybridised with, left panel, CW10; middle panel, JH1. Normal fragments of 41 kb (plus a 31 kb fragment from the 16p13.1 site), CW10, and 18 kb, JHI, are identified with these probes; OX875 has an additional 53 kb band (arrowed). The EcoR I site separating these two fragments is removed by the deletion (see FIG. 3a). The right panel shows a Southern blot of BamH I digested DNA (as above) hybridised with 1A1H.6. A novel fragment of 9.5 kb is seen in OX875 DNA, as well as the normal 15 kb fragment. These results indicate that OX875 has a 5.5 kb deletion; its position was determined more precisely by mapping relative to two Xba I sites which flank the deletion (see FIG. 3a).

FIG. 5b: Northern blot of total fibroblast RNA, as (a), hybridised with the cDNAs, AH4, 3A3 and AH3. A novel transcript (PBP-875) of about 11 kb is seen with AH4 (the band is reduced in intensity because the probe is partly deleted) and AH3 (arrowed), which flank the deletion, but not 3A3 which is entirely deleted (see FIG. 3a). The transcripts HG-A, HG-B and HG-C, from the duplicated area, are;seen with AH3 (see FIG. 4b).

FIG. 5c: Left panel; FIGE of DNA from: normal (N) and ADPKD patient OX114 (114), digested with EcOR I and hybridised with CW10; a novel fragment of 39 kb (arrowed) is seen in OX114. Middle panel; DNA, as above, plus the normal mother (M) and brother (B) of OX114 digested with BamH I and hybridised with CW21. A larger than normal fragment of 19 kb (arrowed) was detected in OX114 but not other family members due to deletion of a BamH I site; together these results are consistent with a 2 kb deletion (see FIG. 3a). Right panel; RT-PCR of RNA, as above, with primers flanking the OX114 deletion (see Experimental Procedures). A novel fragment of 810 bp (arrowed) is seen in OX114, indicating a deletion of 446 bp in the PBP transcript.

FIG. 5d: RT-PCR of RNA from: ADPKD patient OX32 (32) plus the probands, normal mother (M) and affected father (F) and sibs (1) and (2) using the C primer pair from 3A3 (SEQ ID NOS. 11 and 12) (see Experimental Procedures). A novel fragment of 125 bp is detected in each of the affected individuals.

FIG. 6: Map of the region containing the TSC2 and PBP genes showing the area deleted in patient WS-53 and the position of the 77 translocation breakpoint. Localisation of the distal end of the WS-53 deletion was described (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and we have now localised the proximal end between SM6 and JH17. The size of the aberrant Mlu I fragment in WS-53, detected by JH1 and JH17, is 90 kb and these probes lie on adjacent Mlu I fragments of 120 kb and 70 kb, respectively. Therefore the WS-53 deletion is about 100 kb. Restriction sites for: Mlu I (M); Nru I (R) Not I (N); and partial maps for Sac II (S) and BssH II (H) are shown. DNA probes (open boxes) and the TSC2 and PBP transcripts (filled boxes) are indicated below the line with their known genomic extents (brackets). The locations of the microsatellites KG8 and SM6 are also indicated.

FIG. 7: The partial nucleotide sequence (cDNA) of the PKD1 transcript extending 5631 bp to the 3′ end of the gene (SEQ ID NO. 1). The corresponding predicted protein (SEQ ID NO.2) is shown below the sequence and extends from the start of the nucleotide sequence. The GT-repeat, KG8, is in the 3′ untranslated region between 5430-5448 bp. This sequence corresponds to GenBank Accession No. L33243. Also shown is probe 1A1H0.6 [Seq. I.D. No. 3].

FIG. 8: The sequence of the probe 1A1H.0.6 [Seq. I.D. No. 19].

FIG. 9: The sequence of the probe CW10 [Seq I.D. No. 4] which is about 0.5 kb. Also shown are the sequences of probes CW10F [Seq. I.D. No. 10] and CW10R [Seq. I.D. No. 21].

FIG. 10: Panels 10A-10A-KK show the larger partial nucleotide sequence (SEQ ID NO:1) of the PKD1 transcript (cDNA) extending from bp 2 to 13807 bp to the 3′ end of the gene together with corresponding predicted protein (also shown in SEQ ID NO:2). This larger partial sequence encompasses the (smaller) partial sequence of FIG. 7 from amino acid residue 2726 in SEQ ID NO:3 and relates to the entire PKD1 gene sequence apart from its extreme 5′ end.

FIG. 11: A map of the 75 bp intron amplified by the primer set 3A3C [Seq. I.D. Nos. 11 and 12], insert [Seq. I.D. No. 18] at position 3696 of the 3′ sequence [Seq. I.D. No. 1] showing the positions of genomic deletions found in PKD1 patients 461 and OX1054.

FIG. 12: A map of the region of chromosome 16 containing the TSC2 and PKD1 genes showing the areas affected in patients WS-215, WS-250, WS-212, WS-194, WS-227 and WS-219; also WS-53 (but cf. FIG. 6). Genomic sites for the enzymes Mlul (M), Clal (C), Pvul (P) and Nrul (R) are shown. Positions of single copy probes and cosmids used to screen for deletions are shown below the line which represents about 400 kb of genomic DNA. The genomic distribution of the approximately 45 kb TSC2 gene and known extent of the PKD1 gene are indicated above. The hatched area represents an about 50 kb region which is duplicated more proximally on chromosome 16p.

FIG. 13 is a: genomic map of the PKD1 gene. (Top) A restriction map of the genomic area containing the PKD1 gene showing sites for Bam H1(B), EcoRI(E) and partial maps for Xbal (X) and Hind III(H), and the duplicated area (hatched). The position of genomic clones and the cosmid JH2A are shown above the map (open boxes). The positions of the 46 exons of the PKD1 gene are shown below the map (solid boxes, translated areas; open boxes, untranslated regions; UTRs). Each 5th exon is numbered and the direction of transcription arrowed. The area sequenced in FIGS. 7 and 10 is bracketed and the approximate location of the 3′ end of the TSC2 gene is shown on the left (dashed line and hatched box). (Bottom) The cDNA contig covering the PKD1 transcript. The cDNAs are: 1, revl; 2, S13;3, S3/4; 4, S1/3;5, GAP e; 6, GAP d; 7, GAP g; 8, GAP a (see table 2 for details); 9, A1C; 10, AH3; 11, 3A3; 12, AH4.

FIG. 14(a) (Top): Map of the genomic BamH I fragment, SM3 which contains the CpG island at the 5′ end of the PKD1 gene, showing the probe CW45 (open box). Genomic restriction sites for the methylation sensitive enzymes: SacII (S), Notl (N), Mlul (M) and BssHII (H) are illustrated. The approximate position of the DNase1 hypersensitive site is also shown (large arrow), plus the location of the first exon including the proposed transcription start site (small arrow), the 5′UTR (open box) and the translated region (solid bar). (Bottom) The GC content across the area is plotted with a window size of 50 nt. A peak of GC content of over 80% is seen in the area of the transcriptional start site and the first exon. A corresponding lack of CpG suppression was also found with an average CpG/GC ratio of 0.84 between 800-1,800 bp.

FIG. 14(b). Analysis of DNase I hypersensitivity at the PKD1 CpG island. DNA isolated from HeLa cells treated with an increasing amount of DNase I (left to right; first lane contains no DNase 1), digested with BamH I and hybridised with CW45. A fragment about 400 bp smaller than the restriction fragment is seen with increasing DNase 1, indicating a hypersensitive site as shown in (a). SM3 is within the duplicated area and so both the PKD1 and HG loci are assayed together. The degree of DNase1 digestion seen at the end of the assay indicates that cleavage occurs at the PKD1 and HG loci.

FIG. 15 provides the sequence of the PKD1 transcript [Seq. I.D. No. 7] and predicted protein [Seq. I.D. No. 8]. The full sequence of 14,148 bp from the transcription start site to the poly A tail is shown. The probable signal sequence of 23 amino acids is shown after the first methionine (underlined) plus the cleavage site (arrow). The predicted transmembrane (TM) domains (double underlined and numbered) and N-linked glycosylation sites (asterisk) are indicated. The position of a possible hinge sequence is underlined and tyrosine kinase and protein kinase C phosphorylation sites marked with a box and circle, respectively.

FIG. 16(a). The leucine rich repeats (LRRs) found in the PKD1 protein (72-125aa) are compared with each other and to the LRR consensus (Rothberg, 1990; Kobe, 1994); a, aliphatic. A total of just over 2 full repeats are present in PKD1 but they have been arranged into 3 incomplete repeats to show their similarity to those found in slit (Rothberg, 1990). The black boxes show identity to the LRR consensus and shaded boxes other regions of similarity between the repeats which have also been noted in other LRRs (Kobe, 1994).

FIG. 16(b). The amino flanking region to the LRR in the PKD1 protein (33-71aa) is compared similar regions from a variety of other proteins. Black boxes shown identity with the consensus (adapted from [Rothberg, 1990 #1126]) and shaded boxes conserved amino acids. The different types of residue indicated in the consensus are: a, as above; p, polar or turn-like; h, hydrophobic. The listed proteins, with the species and Protein Identification Resource no. (PIR) shown in brackets, are: OMgp, oligodendrocyte myelin glycoprotein (Human, A34210); Slit (Drosophila; A36665); Chaoptin (Drosophila; A29943); GP-IB Beta, platelet glycoprotein 1bβ chain (Human; A31929); Pg1, proteoglycan-1 (mouse; 520811); Biglycan (Human; A40757); Trk (Human; A25184) and LH-CF, lutropinchoriogonadotrophin receptor (Rat; A41343).

FIG. 16(c). The carboxy flanking region of the LRR repeat from the PKD1 protein (126-180 aa) compared to similar regions in other proteins and a consensus accepted from [Rothberg, 1990 #1126]. The shading and amino acid types are as above. The proteins not described above are: Toll (Drosophila; A29943) and GP IX, platelet glycoprotein IX (Human; A46606).

FIG. 17 is a sequence comparison of the C-type lectin domain. The PKD1 lectin domain (403-532aa) is compared to those of: BRA3, acorn barnacle lectin (JC1503); Kupffer cell carbohydrate-binding receptor (Rat; A28166), CSP, cartilage specific protoglycan (Bovine; A27752); Agp; asialoglycoprotein receptor (Human; 55283), E-Selectin (Mouse; B42755) and glycoprotein gp120 (Human; A46274). Black squares show identify with the consensus and shaded boxes conserved residues. Amino acid types are: Very highly conserved residues are shown in bold in the consensus which is adapted from Drickamer 1987, Drickamer 1988.

FIG. 18 is a sequence analysis of the Ig-like repeat. The 16 copies of the PKD1 Ig-like repeat (PKDI 273-356 aa; PKDII-XVI, 851-2145aa) are compared to each other and to: V.a. colAi, and C.p. colA collagenases of Vibrio alginolyticus (S19658) and Clostridium perfringens (D13791), respectively; Pmel17, melanocyte specific glycoprotein (Human; A41234), FLT4, Ig repeat IV of fms-like tyrosine kinase 4 (Human; X68203), CaVPT, Ig repeat I of target protein of the calcium vector protein (CAVP) (amphioxius; P05548). black boxes shown amino acids identical in more than 5 repeats and shaded boxes related residues. An Ig consensus determined from Harpaz et al. 1994 and Takagi et al. 1990 is shown in the symbols: a, aliphatic; h, hydrophobic; s, small and b, base with the predicted positions of the β-strands indicated below. The PKD repeat IV has an extra repetition of 20 aa in the centre of the repeat while all of the others are between 84-87 aa.

FIG. 19 reveals type III-related fibronectin domains. The four fibronectin-related domains from the PKD1 protein (2169-2573aa) are compared to similar domains in: Neuroglian (Drosophila; A32579); L1, neural recognition molecule L1 (X59847); F11, neural cell recognition molecule F11 (X14877); TAG 1, transiently expressed axonal surface glycoprotein-1 (Human; S28830); F3, Neuro-1 antigen (mouse; SO5944); NCAM, neural cell adhesion molecule (Rat; X06564); DCC, deleted in colorectal cancer (Human; X76132); LAR, Leukocyte-common antigen related molecule (Human; YOO815); HPTP, β protein tyrosine phosphate beta (Human; X54131) and FN, fibronectin (Human; X02761). The consensus sequence is compiled from Borh and Doolittle (1993), Kuma et al. (1993), Baron et al. (1992) and Borh and Doolittle (1992). Black boxes show identity to highly conserved residues and shaded boxes conserved changes or similarity in less highly conserved positions. The approximate positions of the β strands are illustrated. The fibronectin repeats in the PKD1 protein are linked by sequences of 27aa (A-B), 22aa (B-C) and 7aa (C-D) which are not shown.

FIG. 20 presents a proposed model of the PKD1 protein, polycystin. The predicted structure of the PKD1 protein is shown.

DETAILED DESCRIPTION

All references mentioned herein are listed in full at the end of the description which are herein incorporated by reference in their entirety. Except where the context clearly indicates otherwise, references to the PBP gene, transcript, sequence, protein or the like can be read as referring to the PKD1 gene, transcript, sequence, protein or the like, respectively.

A Translocation Associated with ADPKD

A major pointer to the identity of the PKD1 gene was provided by a Portuguese pedigree (family 77) with both ADPKD and TSC (FIG. 1b). Cytogenetic analysis showed that the mother, 77-2, has a balanced translocation, 46XX t(16;22) (p13.3;q11.21) which was inherited by her daughter, 77-3. The son, 77-4, has the unbalanced karyotype, 45XY-16-22+der(16) (16qter-->16p13.3:22q11.21-->2qter) and consequently is monosomic for 16p13.3-->16pter as well as for 22q11.21-->22pter. This individual has the clinical phenotype of TSC (see Experimental Procedures); the most likely explanation is that the TSC2 locus located within 16p13.3 is deleted in the unbalanced karyotype.

Further analysis revealed that the mother (77-2), and the daughter (77-3) with the balanced translocation, have the clinical features of ADPKD (see Experimental Procedures), while the parents of 77-2 were cytogenetically normal, with no clinical features of TSC and no renal cysts on ultrasound examination (aged 67 and 82 years). Although kidney cysts can be a feature of TSC, no other clinical signs of TSC were identified in 77-2 or 77-3, making it unlikely that the polycystic kidneys were due to TSC. We therefore investigated the possibility that the translocation disrupted the PKD1 locus in 16p13.3 and proceeded to identify and clone the region containing the breakpoint.

The 77 family was analyzed with polymorphic markers from 16p13.3. Individual 77-4 was hemizygous for MS205.2 and GGG1, but heterozygous for SM6 and more proximal markers, locating the translocation breakpoint between GGG1 and SM6 (see FIG. 1a). Fluorescence in situ hybridization (FISH) of a cosmid from the TSC2 region, CW9D (cosmid 1 in FIG. 1a), to metaphase spreads showed that it hybridized to the der(22) chromosome of 77-2; placing the breakpoint proximal to CW9D and indicating that 77-4 was hemizygous for this region consistent with his TSC phenotype. DNA from members of the 77 family was digested with Cla I, separated by PFGE and hybridized with SM6; revealing a breakpoint fragment of about 100 kb in individuals with the der(16) chromosome (FIG. 1c). The small size of this novel fragment enabled the breakpoint to be localized distal to SM6 in a region of just 60 kb (FIG. 1a). A cosmid contig covering this region was therefore constructed (see Experimental Procedures for details).

The Translocation Breakpoint Lies within a Region Duplicated Elsewhere on Chromosome 16p (16p13.1)

It is noted hereabove that the region between CW21 and N54 (FIG. 1a) was duplicated at a more proximal site on the short arm of chromosome 16 (Germino, et al., 1992; European Chromosome 16 Tuberous Sclerosis Consortium, 1993). FIG. 2 shows that a cosmid, CW10III, from the duplicated region hybridized to two points on 16p; the distal, PKD1 region and a proximal site positioned in 16p13.1. The structure of the duplicated area is complex with each fragment present once in 16p13.3 re-iterated two-four times in 16p13.1 (see FIG. 2). Cosmids spanning the duplicated area in 16p13.3 were subcloned (see FIG. 3a and Experimental Procedures for details) and a restriction map was generated. A genomic map of the PKD1 region was constructed using a radiation hybrid, Hy145.19 which contains the distal portion of 16p but not the duplicate site in 16p13.1.

To localize the 77 translocation breakpoint, subclones from the target region were hybridized to 77-2 DNA, digested with Cla I and separated by PFGE. Once probes mapping across the breakpoint were identified they were hybridized to conventional Southern blots of 77 family DNA. FIG. 3b shows that novel BamH I fragments were detected from the centromeric and telomeric side of the breakpoint, which was localized to the distal part of the probe 8S1 (FIG. 3a). Hence, the balanced translocation was not associated with a substantial deletion, and the breakpoint was located more than 20 kb proximal to the TSC2 locus (FIG. 3a). These results supported the hypothesis that polycystic kidney disease in individuals with the balanced translocation (77-2 and 77-3) was not due to disruption of the TSC2 gene, but indicated that a separate gene mapping just proximal to TSC2, was likely to be the PKD1 gene.

The Polycystic Breakpoint (PBP) Gene is Disrupted By the Translocation

Localization of the 77 breakpoint identified a precise region in which to look for a candidate or the PKD1 gene. During the search for the TSC2 gene we identified other transcripts not associated with TSC including a large transcript (about 14 kb) partially represented in the cDNAs 3A3 and AH4 which mapped to the genomic fragments CW23 and CW21 (FIG. 3a). The orientation of the gene encoding this transcript had been determined by the identification of a polyA tract in the cDNA, AH4: the 3′ end of this gene lies very close to the TSC gene, in a tail to tail orientation (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). To determine whether this gene crossed the translocation breakpoint genomic probes from within the duplicated area and flanking the breakpoint were hybridized to Northern blots. Probes from both sides of the breakpoint, between JH5 and JH13 identified the 14 kb transcript (FIG. 3a and see below for details). Therefore, this gene, called 3A3 , but not designated the PBP gene extended over the 77 breakpoint and consequently was a candidate for the PKD1 gene. A walk was initiated to increase the extent of the PBP cDNA contig and several new cDNAs were identified using probes from the single copy (non-duplicated) region (see Experimental Procedures for details). A cDNA contig was constructed which extended about 5.7 kb, including about 2 kb into the area that is duplicated (FIG. 3a).

Expression of the PBP Gene

Initial studies of the expression pattern of the PBP gene were undertaken with cDNAs that map entirely within the single copy region (e.g. AH4 and 3A3). FIG. 4a shows that the about 14 kb transcript was identified by 3A3 in various tissue-specific cell lines. From this and other Northern blots we concluded that the PBP gene was expressed in all of the cell lines tested, although often at a low level. The two cell lines which showed the highest level of expression were fibroblasts and a cell line derived from an astrocytoma, G-CCM. Significant levels of expression were also obtained in cell lines derived from kidney (G401) and liver (Hep3B). Measuring the expression of the PBP gene in tissue samples by Northern blotting proved difficult because such a large transcript is susceptible to minor RNA degradation. However, initial results with an RNAse protection assay, using a region of the gene located in the single copy area (see Experimental Procedures), showed a moderate level of expression of the PBP gene in tissue obtained from normal and polycystic kidney (data not shown). The widespread expression of the PBP is consistent with the systemic nature of ADPKD.

Identification of Transcripts that are Partially Homologous to the PBP Transcript

New cDNAs were identified with the genomic fragments, JH4 and JH8, that map to the duplicated region (FIG. 3a and see Experimental Procedures). However, when these cDNAs were hybridized to Northern blots a more complex pattern than that seen with 3A3 was observed. As well as the ˜14 kb PBP transcript, three other, partially homologous transcripts were identified designated homologous gene-A (HG-A; ˜21 kb), HG-B (˜17 kb) and HG-C (8.5 kb) FIG. 4b). There were two possible explanations for these results, either the HG. transcripts were alternatively spliced forms of the PBP gene, or the HG transcripts were encoded by gene located in 16p13.1. To determine the genomic location of the HG loci a fragment from the 3′ end of one HG cDNA (HG-4/1.1) was isolated. HG-4/1.1 hybridized to all three HG transcripts, but not to the PBP transcript and on a hybrid panel it mapped to 16p13.1 (not the PKD1 area). These results show that all the HG transcripts are related to each other outside the region of homology with the PBP transcript and that the HG loci map to the proximal site (16p13.1).

An Abnormal Transcript Associated with the 77 Translocation

As the PBP gene was transcribed across the region disrupted by the 77 translocation breakpoint, in a proximal to distal direction on the chromosome (see FIG. 3a) it was possible that a novel transcript originating from the PBP promotor would be found in this family. FIG. 4c shows that using a probe to the PBP transcript that mapped mainly proximal to the breakpoint, a novel transcript of approximately 9 kb (PBP-77) derived from the der(16) product of the translocation was detected. Interestingly, the PBP-77 transcript appears to be expressed at a higher level than the normal PBP product. These results confirmed that the 77 translocation disrupts the PBP gene and supports the hypothesis that this is the PKD1 gene.

Mutations of the PBP Gene in Other ADPKD Patients

To prove that the PBP gene is the defective gene at the PKD1 locus, we analyzed this region for mutations in patients with typical ADPKD. The 3′ end of the PBP gene was most accessible to study as it maps outside the duplicated area. To screen this region BamH I digests of DNA from 282 apparently unrelated ADPKD patients were hybridized with the probe 1A1H.6 [Seq. I.D. No. 3], (see FIG. 3a). In addition, a large EcoR I fragment (41 kb) which contains a significant proportion of the PBP gene was assayed by field inversion gel electrophoresis (FIGE) in 167 ADPKD patients, using the probe CW10 [Seq. I.D. No. 4]. Two genomic rearrangements were identified in ADPKD patients by these procedures; each identified by both methods.

The first rearrangement was identified in patient OX875 (see Experimental Procedures for clinical details) who was shown to have a 5.5 kb genomic deletion without the 3′ end of the PBP gene, producing a smaller transcript (PBP-875) (see FIGS. 5a, b and 3 a for details). This genomic deletion results in a ˜3 kb internal deletion of the transcript with the ˜500 bp adjacent to the polyA tail intact. In this family linkage of ADPKD to chromosome 16 could not be proven because although OX875 has a positive family history of ADPKD there were no living, affected relatives. However, paraffin-embedded tissue from her affected father (now deceased) was available. We demonstrated that this individual has the same rearrangement as OX875 by PCR amplification of a 220 bp fragment spanning the deletion (data not shown). This result and analysis of two unaffected sibs of OX875, that did not have the deletion, showed that this mutation was transmitted with ADPKD.

The second rearrangement detected by hybridization was a 2 kb genomic deletion within the PBP gene, in ADPKD patient OX114 (see Experimental Procedures for clinical details and FIGS. 5c and 3 a). No abnormal PBP transcript was identified by Northern blot analysis, but using primers flanking the deletion (see Experimental Procedures) a shortened product was detected by RT-PCR (FIG. 5c). This was cloned and sequenced and shown to have a frame-shift deletion of 446 bp (between base pair 1746 and 2192 of the sequence shown in FIG. 7 [Seq. I.D. No. 1]). OX114 is the only member of the family with ADPKD (she has no children) and ultrasound analysis of her parents at age 78 (father) and 73 years old (mother) showed no evidence of renal cysts. Somatic cell hybrids were produced from OX114 and the deleted chromosome was found to be of paternal origin by haplotype analysis. The father of OX114 (OX984) with seven microsatellite markers from the PKD1 region, as OX114. Renal ultrasound revealed no cysts in OX984 at age 53 and no deletion was detected by DNA analysis (FIG. 5c). Hence, the deletion in OX114 is a de novo event associated with the development of ADPKD. Although it is not possible to show that the ADPKD is chromosome 16-linked, the location of the PBP gene indicated that this is a de novo PKD1 mutation.

To identify more PKD1 associated mutations, single copy regions of the PBP gene were analyzed by RT-PCR using RNA isolated from lymphoblastoid cell lines established from ADPKD patients. cDNA from 48 unrelated patients was amplified with the primer pair 3A3 C [Seq. I.D. Nos. 11 and 12] (see Experimental Procedures) and the product of 260 bp was analyzed on an agarose gel. In one patient, OX32, an additional smaller product (125 bp) was identified, consistent with a deletion or splicing mutation. OX32 comes from a large family in which the disease can be traced through three generations. Analysis of RNA from two affected sibs of OX32 and his parents showed that the abnormal transcript segregates with PKD1 (FIG. 5d).

Amplification of normal genomic DNA with the 3A3 C primers (Seq. I.D. Nos. 11 and 12) generates a product of 418 bp; sequencing showed that this region contains two small introns (5′, 75 bp and 3′, 83 bp) flanking a 135 bp exon. The product amplified from OX32 genomic DNA was normal in size, excluding a genomic deletion. However, heteroduplex analysis of that DNA revealed larger heteroduplex bands, consistent with a mutation within that genomic interval. The abnormal OX32, RT-PCR product was cloned and sequenced: this demonstrated that, although present in genomic DNA, the 135 bp exon was missing from the abnormal transcript. Sequencing of OX32 genomic DNA demonstrated a G-->C transition at +1 of the splice donor site following the 135 bp exon. This mutation was confirmed in all available affected family members by digesting amplified genomic DNA with the enzyme Bst NI: a site is destroyed by the base substitution. The splicing defect results in an in-frame deletion of 135 bp from the PBP transcript (3696 bp to 3831 bp of the sequence shown in FIG. 7 [Seq. I.D. No. 1]). Together, the three intragenic mutations confirm that the PBP gene is the defective gene at the PKD1 locus.

Deletions that Disrupt the TSC2 and the PKD1 Gene

The deletion called WS-53 disrupts both the TSC2 gene and the PKD1 gene (European Chromosome 16 Tuberous Sclerosis Consortium, 1993), although the full proximal extent of the deletion was not determined. Further study has shown that the deletion extends ˜100 kb (see FIG. 6 for details) and deletes most if not all of the PKD1 gene. This patient has TSC but also has unusually severe polycystic disease of,the kidneys. Other patients with a similar phenotype have also been under investigation. Deletions involving both TSC2 and PKD1 were identified and characterized in six patients in whom TSC was associated with infantile polycystic kidney disease. As well as the deletion in WS-53, those in WS-215 and WS-250 also extended proximally well beyond the known distribution of PKD1 and probably delete the entire gene. The deletion in WS-194 extended over the known extent of PKD1, but not much further proximally, while the proximal breakpoints in WS-219 and WS-227 lay within PKD1 itself. Northern analysis of case WS-227 lay within PKD1 itself. Northern analysis of case WS-219 with probe JH8, which lies outside the deletion, showed a reduced level of the PKD1 transcript but no evidence of an abnormally sized transcript (data not shown). Analysis of samples from the clinically unaffected parents of patients WS-53, WS-215, WS-219, WS-227 and WS-250 showed the deletions in these patients to be de novo. The father of WS-194 was unavailable for study.

In a further case (WS-212), renal ultrasound shoed no cysts at four years of age but a deletion was identified which removed the entire TSC2 gene and deleted an XbaI site which is located 42 bp 5′ to the polyadenylation signal of PKD1. To determine the precise position of the proximal breakpoint in PKD1, a 587 bp probe from the 3′ untranslated region (3′UTR) was hybridized to XbaI digested DNA. A 15 kb Xbal breakpoint fragment was detected with an approximately equal intensity to the normal fragment of 6 kb, indicating that most of the PKD13′VTR was preserved on the mutant chromosome. Evidence that a PKD1 transcript is produced from the deleted chromosome in WS-212 was obtained by 3′ rapid identification of cDNA ends (RACE) with a novel, smaller product generated from WS-212 cDNA. Characterization of this product showed that polyadenylation occurs 546 bp 5′ to the normal position, within the 3′UTR of PKD1 (231 bp 3′ to the stop codon at 5073 bp of the described PKD1 sequence¹⁴ [Seq. I.D. No. 1]). A transcript with an intact open reading frame is thus produced from the deleted WS-212 chromosome. It is likely that a functional PKD1 protein in produced from this transcript, explaining the lack of cystic disease in this patient. The sequence preceding the novel site of polyA addition is: AGTCAGTAATTTATATGGTGTTAAAATGTG(A)n [Seq. I.D. No. 22]. Although not conforming precisely to the consensus of AATAAA, it is likely that part of this AT rich region acts as an alternative polyadenylation signal if, as in this case, the normal signal is deleted (a possible sequence is underlined).

The WS-212 deletion is 75 kb between SM9-CW9 distally and the PKD1 3′UTR proximally. The WS-215 deletion is 160 kb between CW15 and SM6-JH17. WS-194 has 65 kb deleted between CW20 and CW10-CW36. WS-227 has a 50 kb deletion between CW20 and JH11 and WS-219 has a 27 kb deletion between JH1 and JH6. The distal end of the WS-250 deletion is in CW20 but the precise location of the proximal end is not known. However, the same breakpoint fragment of 320 kb is seen with Pvul-digested DNA using probes on adjacent Pvul fragments, CE18 (which normally detects a 245 kb fragment) and Blu24 (235 kb). Hence this deletion can be estimated ˜160 kb. b. PFGE analysis of the deletion in WS-219. Mlul digested DNA from a normal control (N) and WS-219 probed with the clones H2, JH1, CW21 and CW10 [Seq. I.D. No. 4] which detect an ˜130 kb fragment in normal individuals. CW10 [Seq. I.D. No. 4] also detects a much smaller fragment from the duplicated region situated more proximally on 16p. A novel fragment of 100 kb is seen in WS-219 with probes H2 and CW10 [Seq. I.D. No. 4] which flank the deletion in this patient. JH1 is partially deleted but detects the novel band weakly. The aberrant fragment is not detected by CW-21, which is deleted on the mutant chromosome. BamHl digested DNA of normal control (N) and WS-219 separated by conventional gel electrophoresis and hybridized to probes JH1 and JH6 which flank the deletion. The same breakpoint fragment of ˜3 kb is seen with both probes, consistent with a deletion of ˜27 kb ending within the BamHl fragments seen by these probes.

Two Further Deletions

In addition we have characterized two further mutations of this gene which were identified in typical PKD1 families. In both cases the mutation is a deletion in the 75 bp intron amplified by the primer pair 3A3C [Seq. I.D. Nos. 11 and 12] (European Polycystic Kidney Disease Consortium, 1994). The deletions are of 18 bp and 20 bp, respectively, in the patients 461 and OX1054. Although these deletions do not disrupt the highly conserved sequences flanking the exon/intron boundaries, they do result in aberrant splicing of the transcript. In both cases, two abnormal mRNAs are produced, one larger and one smaller than normal. Sequencing of these cDNAs showed that the larger transcript includes the deleted intron, and so has an in-frame insertion of 57 bp in 461, while OX1054 has a frameshift insertion of 55 bp. The smaller transcript is due to activation of a cryptic splice site in the exon preceding the deleted intron and results in an in-frame deletion of 66 bp in both patients. The demonstration of two additional mutations of this gene in PKD1 patients further confirms that this is the PKD1 gene.

Partial Characterization of the PKD1 Gene

To characterize the PKD1 gene further, evolutionary conservation was analyzed by ‘zoo blotting’. Using probes from the single copy, 3′ region (3A3) and from the duplicated area (JH4, JH8) the PKD1 gene was conserved in other mammalian species, including horse, dog, pig and rodents (data not shown). No evidence of related sequences were seen in chicken, frog or drosophila by hybridization at normal stringency. The degree of conservation was similar when probes from the single copy of the duplicated region were employed.

Although the full genomic extent of the PKD1 gene was not yet known, results obtained by hybridization to Northern blots showed that it extended from at least as far as JH13. Several CpG islands were localized 5′ of the known extent of the PKD1 gene (FIG. 6), although there was no direct evidence that any of these are associated with this gene.

The cDNA contig extending 5631 bp to the 3′ end of the PKD1 transcript was sequenced; where possible more than one cDNA was analyzed and in all regions both strands were sequenced (FIG. 7 [Seq. I.D. No. 1]). We estimated that this accounts for ˜40% of the PKD1 transcript. An open reading frame was detected which runs from the 5′ end of the region sequenced and spans 4842 bp, leaving a 3′ untranslated region of 789 bp which contains the previously described microsatellite, KG8 (Peral, et al., 1994; Snarey, et al., 1994). A polyadenylation signal is present at nucleotides 5598-5603 and a polyA tail was detected in two independent cDNAs (AH4 and AH6) at position, 5620. Comparison with the cDNAs HG-4 and 11BHS21, which are encoded by genes in the duplicate, 16p13.1 region, show that 1866 bp at the 5′ end of the partial PKD1 sequence shown in FIG. 7 [Seq. I.D. No. 1] lies within the duplicated area. The predicted amino acid sequence from the available open reading frame extends 1614 residues, and is shown in FIG. 7 [Seq. I.D. No. 1]. A search of the swissprot and NBRF data bases with the available protein sequence, using the Blast program (Altschul, et al., 1990) identified only short regions of similarity (notably, between amino-acids 690-770 and 1390-1530) to a diverse group of proteins; no highly significant areas of homology were recognized. The importance of the short regions of similarity. is unclear as the search for protein motifs with the ProSite Program did not identify any recognized functional protein domains within the PKD1 gene.

The test of identifying and characterizing the PKD1 gene has been more difficult than for other disorders because more than three quarters of the gene is embedded in a region of DNA that is duplicated elsewhere on chromosome 16. This segment of 40-50 kb of DNA, present as a single copy in the PKD1 area (16p13.3), is reiterated as several divergent copies in the more proximal region, 16p13.1. This proximal site contains three gene loci (HG-A, -B and -C) that each produce polyadenylated mRNAs and share substantial homology to the PKD1 gene; it is not known whether these partially homologous transcripts are translated into functional proteins.

Although gene amplification is known as a major mechanism for creating protein diversity during evolution, the discovery of a human disease locus embedded within an area duplicated relatively recently is a new observation. In this case because of the recent nature of the reiteration the whole duplicated genomic region retains a high level of homology, not just the exons. The sequence of events leading to the duplication and which sequence represents the original gene locus are not yet clear. However, early evidence of homology of the 3′ ends of the three HG transcripts which are different from the 3′ end of the PKD1 gene indicated that the loci in 16p13.1 have probably arisen by further reiteration of sequences at this site, after it separated from the distal locus.

To try to overcome the duplication problem we employed an exon linking approach using RNA isolated from a radiation hybrid, HY145.19, that contains just the PKD1 part of chromosome 16, and not the duplicate site in 16p13.1. Hence, this hybrid produces transcripts from the PKD1 gene but not from the homologous genes (HG-A, HG-B and HG-C). We have also sequenced much of the genomic region containing the PKD1 gene, from the cosmid JH2A, and have sequenced a number of cDNAs from the HG locus. To determine the likely position of PKD1 exons in the genomic DNA we compared HG cDNAs, (HG-4 and HG-7) to the genomic sequence. We then designed primers with sequences corresponding to the genomic DNA, to regions identified by the HG exons and employing DNA generated from the hybrid HY145.19, we amplified sections of the PKD1 transcript. The polymerase Pfu was used to minimise incorporation errors. These amplified fragments were then cloned and sequenced. The PKD1 cDNA contig whose sequence is shown in FIG. 10 is made up of (3′-5′) the original 5.7 kb of sequence shown in FIG. 7, and the cDNAs: gap α 22 (890 bp), gap gamma (872 bp), a section of genomic DNA from the clone JH8 (2,724 bp) which corresponds to a large exon, S1-S3 (733 bp), S3-S4 (1,589 bp) and S4-S13 (1,372 bp). Together these make a cDNA of 13,807 nt. When these cDNAs from the PKD1 contig were sequenced an open reading frame was found to run from the start of the contig to the stop codon, a region of 13,018 bp (SEQ ID NO. 5). The predicted protein encoded by the PKD1 transcript is also shown in FIG. 10 (SEQ ID NO. 6) and has 4,339 amino acid residues.

Cloning a Full Length PKD1 cDNA

cDNAs known to originate from the PKD1 or HG transcripts show on average a sequence divergence of less than 3%. Consequently, although many cDNAs were identified by hybridisation of various PKD1 genomic probes to cDNA libraries, it proved difficult to differentiate genuine PKD1 clones from those of the HG transcripts. For this reason a novel strategy was employed to clone the PKD1 transcript.

To obtain a template of genomic sequence of the PKD1 gene, clones which contain the transcribed region, JH6 and JH8-JH13, were sequentially truncated and sequenced. These clones were isolated from the cosmid JH2A, which extends into the single copy area containing the 3′ portion of the PKD1 gene (FIG. 13) and hence represents the PKD1 and not the HG loci. As a result of this analysis a contig of about 18 kb of genomic sequence was generated, which was ultimately found to encode >95% of the unsequenced portion of the PKD1 transcript.

A number of HG cDNA clones identified by the DNA probes JH8 or JH13 (including HG-4, HG-7C and 13A1) were sequenced. Clones identified by JH8 were chosen because this genomic area is duplicated fewer times than the surrounding DNA, with only the HG-A and HG-B transcripts (not HG-C) homologous to this region. The comparison of these cDNA and genomic sequences showed a characteristic intron/exon pattern and we concluded that the exons highlighted in the genomic sequence were likely to be exons of the PKD1 gene. To prove this, pairs of primers matching the sequence of the putative PKD1 exons and spaced 0.7-2 kb apart in the proposed transcript, were synthesised. Employing RNA from a radiation hybrid, HY145.19, that contains the PKD1 but not the HG loci, PKD1 specific cDNAs were amplified by RT-PCR and cloned (see Experimental Procedures for details). In this way, a number of overlapping cDNAs spanning the PKD1 transcript, for the cDNAs at the 3′ end to those homologous to JH13 were cloned (FIG. 13).

Analysis of a further cDNA, HG-6 showed that a short region (−100 bp) of HG-6 lay 5′ to the sequenced genomic region and this was located by hybridisation to the genomic clone SM3 (FIG. 13); SM3 was subsequently sequenced. The position of the cDNA in SM3 was identified and the possible 5′ extent of this exon was determined in the genomic sequence; and in-frame stop codon was identified hear the 3′ end of the exon. This exon lay at a CpG island (described hereinafter) suggesting, along with the presence of the stop codon, that this may be the first exon of the PKD1 gene. to determine the likely transcriptional start site the method of primer extension from three different oligos within the first exon was employed (see Experimental Procedures). In all cases, a transcriptional start was identified at the same G nucleotide and showed the first exon to be 426 bp. The structure of the PKD1 transcript was confirmed by a final exon link, rev1 which starts 3 bp 3′ to the proposed transcriptional start (see FIG. 13 and Experimental Procedures for details).

The Intron/exon Structure of the PKD1 Gene

Sequencing the cDNA contig revealed a total sequence of 14, 148 bp which extends over approximately 52 bp of genomic sequence from SM3 to BFS5 (FIG. 13). We were able to determine the intron/exon structure of much of the gene by direct comparison between the cDNA and genomic sequence. In the 3′ region of the gene (JH5-BFS5), a partial genomic sequence was obtained at intron/exon borders by sequencing the corresponding genomic clone from exonic primer.

The PKD1 CpG Island

The 5′ end of the gene lies at CpG island SM3. SM3 is located entirely within the duplicated region, but this clone was isolated from the cosmid SMll which extends through the duplicated area into the proximal flanking single copy region and therefore is known to originate from this area. FIG. 14 shows a map of the PKD1 CpG island including genomic sites for several methylation sensitive enzymes, the location of the first exon and the GC content across the island. Evidence that the enzyme sites in the PKD1 region (and not just the HG area) digest, was obtained by pulsed field gel electrophoresis with the enzymes Mlul, Notl and BssHll using probes outside the duplicated area. Digestion of the Sacll sites and confirmation of the Notl site was made with a panel of somatic cell hybrids which either contain just the HG (P-MWH2A) or just the PKD1 locus (Hy145.19). These results showed that the Sacll and Notl sites digest in both sets of hybrids (data not shown), indicating that this region is a CpG island in the HG as well as the PKD1 area. Further proof that this is the likely position of a functional promoter was obtained by analysis for DNAase 1 hypersensitivity. A DNAase hypersensitive site in the region 5′ to the transcription start site in SM3 was detected (FIGS. 14a and b).

Analysis of the PKD1 Transcript

Analysis of the sequence shows an open reading frame running from the start of the sequence to position 13,117 bp (FIG. 15 [SEQ ID NO. 7]). Detailed sequencing of the genomic region containing the 3′ portion of the gene revealed two extra Cs at positions 13,081-2 (FIG. 15 (SEQ ID NO. 7)). An in-frame start codon which is consistent with the Kozak consensus was detected at position 212 bp; just 3′ to the stop codon in the 5′UTR. Analysis for a signal sequence cleavage site using the von Hinge (von Hinge 1986) algorithm showed a high probability of a hydrophobic signal sequence with cleavage at amino acid 23 (see FIG. 15 (SEQ ID NO. 8)). The total length of the predicted protein is 4302 aa with a calculated molecular mass after excision of the signal peptide of 460 kD and an estimated isoelectric point of 6.26. However, this may be an underestimate of the total mass of the protein as many potential sites for N-linked glycosylation are present (FIG. 15 (SEQ ID NO.8)).

Homologies with the PKD1 Protein

The predicted PKD1 protein was analysed for homologies with know proteins in the SwissProt and NBRF databases using the BLAST Altschul et al 1990) and FASTA algorithms. This analysis revealed two clear homologies and also a number of other potential similarities which were studied on detail.

Leucine Rich Repeat

Near the 5′ end of the PKD1 protein is a region of leucine rich-repeats (LRRs). LRRs are a highly conserved motif usually of 24 residues with precisely spaced leucines (or other aliphatic amino acids) and an asparagine at position 19 (FIG. 16a and reviewed in Kobe and Reisenhofer (1994)). Two complete LRRs plus a partial repeat unit are found in the PKD1 protein, which have complete homology with the LRR consensus.

Surrounding the LRRs are distinctive cysteine-rich amino and carboxy flanking regions (FIGS. 16b and c). This flank-LRR-flank structure is exclusively found on proteins in extracellular locations and is thought to be involved in protein-protein interactions such as adhesion to other cells or to components of the extracellular matrix or as a receptor concerned with binding or signal transduction. The structure found in the PKD1 protein is similar to that found in the Drosophila protein, slit, which is important for normal central nervous system development (Rothberg, 1990). Although slit contains far more LRRs than the PKD1 protein, with four blocks each consisting of 4 or 5 repeat units, the structure of each block is similar as they finish on the amino and carboxy side with shortened LRRs which are immediately flanked by the cysteine rich regions. In the PKD1 protein two shortened LRRs surround one complete repeat unit and immediately abut the amino and carboxy flanking regions.

The amino flanking region consists of four invariant cysteines and a number of other highly conserved residues in an area of 30-40 amino acids; comparison of the PKD1 region to amino flanking motifs of other proteins is shown in FIG. 4b. The carboxy flanking region extends over an area of between 50-60 residues and consists of an invariant proline and four cysteines plus several other highly conserved amino acids. The similarity of the PKD1 region to carboxy flanking regions from other proteins is shown in FIG. 4c.

Some LRR proteins, such as slit (Rothberg 1990) and small proteoglycans are wholly extracellular but others including Toll (Hashimoto et al, 1990) and trkc (Lamballe 1991) have a single transmembrane sequence, while the LH-CRG receptor and related proteins have seven trans-membrane segments and are involved in signal transduction.

C Type Lectin Domain

Analysis of the sequence from exons 6 and 7 showed a high level of homology with a C type lectin domain. C type lectins are found in a variety of proteins in extracellular locations where they bind specific carbohydrates in the presence of Ca²+ ion (Drickamer 1987, 1988; Weiss 1992) FIG. 17 illustrates the similarity of the PKD1 lectin domain to those found in a number of proteins including: proteogylcans, which interact with collagens and other components of the extracellular matrix; endocytic receptors, and selectins which are involved in cell adhesion and recognition. Three different selectins have been identified: E-selectin (endothelium), P-selectin (platelets) and L-selectin (lymphocytes) and these work with other cell adhesion molecules to promote binding of the cell carrying the selectin to various other target cells.

Immunoglobulin-like Repeat Motif

Significant homologies were detected between a region of exon 5 and three regions of exon 15, with the same conserved sequence, WDFGDGS (SEQ ID NO. 8), which is also found in a melanocyte-specific secreted glycoprotein, Pmel17 (Kwon et al, 1991) and three prokaryotic collagenases or proteinases (Ohara et al, 1989, Takeuchi et al, 1992 and Matsushita et al, 1994). Further analysis of the amino acid sequence of the PKD1 protein showed that a conserved region of approximately 85 bp could be discerned around this central sequence and that 16 copies of this repeat were present in the PKD1 protein; 1 in exon 5 and the other 15 as a tandem array in exons 11 to 15. FIG. 18 shows that a highly conserved structure is maintained between the repeats although in some cases less similarity is noted with the WDFGDGS (SEQ ID NO. 8) sequence. Further analysis of the most conserved residues found in the repeat units showed similarity to various immunoglobulin (Ig) domains; two Ig repeats which show particular homology to the PKD1 protein are shown (FIG. 18). The repeat unit is most similar to that found in a number of cell adhesion and surface receptors which have recently been defined as the I set of Ig domains (Harpaz 1994). Ig repeats consist of 7-9 β strands of 5-10 residues linked by turns which are packed into two β sheets. The B, C, F and G β-strands of the I set are particularly similar to the PKD1 repeat, although the highly conserved cystine residues which stabilise the two β sheets through a disulphide bond are absent. The D and E β strands, however, seem less similar and in some cases are significantly shortened or apparently absent. Further evidence that this PKD1 repeat has an Ig-like structure is found by analysis of the secondary structure with the predominant configuration found of β strands linked by turns. The WDFGDS (SEQ ID NO. 23) area of the Ig molecule is one that often has a specific binding function (Jones et al., 1995) and this sequence may have a specific binding role in polycystin.

Type III Fibronectin-related Domains

Analysis of the secondary structure of the PKD1 protein beyond the carboxy end of the region of lg-like repeats showed a continuation of the β stand and turn structure. No evidence of further Ig-like repeats could be found in this area but three pairs of evenly spaced (38-40aa) tryptophan and tyrosine residues was noted which are the most highly conserved positions of the type III fibronectin repeat which has a similar secondary structure to Ig domains. Further analysis and comparison with other type III fibronectin domains showed that in total four fibronectin repeats (one with leucine replacing the conserved tyrosine) could be recognised in this area with many of the most highly conserved residues of this domain found in the PKD1 repeat (FIG. 20).

A large number of proteins with Ig-like repeats have now been described which are involved in cell-cell interactions and cell adhesion (reviewed in: Brummendork and Rathjen, 1994), while type III fibronectin (FNIII) domains are found on extracellular matrix molecules and adhesion proteins. A number of cell adhesion proteins which are located mainly on neural cells, have both Ig-like and FNIII-related domains. In these cases the FNIII repeats are always positioned C-terminal of the Ig-like units and close to a transmembrane domain; a similar pattern is seen in the proposed structure of polycystin. These Ig/FNIII containing proteins such as neuroglican and NrCAM are thought to be involved in neuron-neuron interactions and the patterning of the axonal network.

Many cell adhesion proteins of the Ig superfamily are also involved in communication and signal transduction mediated through their cytoplasmic tails. These cytoplasmic regions are known to bind to cytoskeletal proteins and other intracellular components, and phosphorylation of this part of the molecule is also thought to affect adhesive properties of the protein; potential phosphorylation sites are found in the cytoplasmic tail and one intracellular loop of polycystin (FIG. 20).

Transmembrane Regions

Analysis of hydrophobicity predicted that the deduced protein is an integral membrane protein with a signal peotide and multiple transmembrane (TM) domains located in the C-terminal region. From this analysis 11 regions (including the signal peptide) had a mean hydrophobicity indice higher than 1.4 and therefore were considered as certain membrane spanning domains (see Experimental Procedures for details). Three others with a mean hydrophobicity indice between 0.75-1.0 were considered as putative TM domains. The most likely topology of the protein was predicted using TopPed II programme (see Experimental Procedures for details) and the resulting model included one putative segment plus the 10 certain transmembrane domains and the signal peptide. According to this model the N-terminal end is extracellular and the (highly hydrophobic) carboxy-terminal region is anchored to the membrane by 11 membrane-spanning segments, with the highly charged carboxy end located in the cytoplasm. This topology is supported by the study of N-glycosylation sites with all but one site, out of a total of 61 predicted, in an extracellular location according to the model, including 11 in the two large extracellular loops between TM regions.

However, if degree of hydrophobicity required to define a certain putative transmembrane region is altered within the model, the predicted number of such domains can change to 9 (excluding the most N-terminal pair) or 13 (with two new domains defined between TM7 and TM8). This can be ascertained by studies with specific antibodies.

Most transmembrane proteins containing the types of cell adhesion domain found on polycystin have a single transmembrane domain. The role of the multiple membrane spanning domains found in polycystin is not yet clear.

Proposed Structure of the PKD1 Protein

From the detailed analysis of the predicted PKD1 protein sequence a model of the likely structure of the protein can be formulated (FIG. 20). This model predicts an extracellular N-terminal region of approximately 2550 aa containing several distinctive extracellular domains and an intracellular C-terminus of approximately 225 aa. The intervening region of nearly 1500 aa is associated with the membrane with 11 transmembrane regions predicted and 10 variously sized extracellular and cytoplasmic loops (see FIG. 20). A proline rich hinge is found between the flank-LRR-flank region and the first Ig-like repeat. Two phosphorylation sites for tyrosine kinase and protein kinase C are found in cytoplasmic locations (FIGS. 15 (SEQ ID NO. 8) and 20).

Therefore, the PKD1 protein, named polycystin, has highlighted several clear domains, plus a reiterated motif that occupies over 30% of the protein.

Characterisation of the PKD1 gene has proven to be a uniquely difficult problem because most of the gene lies in a region which is reiterated elsewhere on the chromosome. The high degree of similarity between the two areas (>97%) both in exons and introns has meant that a novel approach has been required to clone the full length transcript; involving extensive genomic sequencing and generating cDNAs from a cell line with the PKD1 but not the HG loci. In this way a contig containing the entire PKD1 transcript has now been cloned.

Preliminary analysis shows that the HG genes are very similar to PKD1 both in terms of genomic structure and sequence over most of their length (apart from the novel 3′ regions). The 5′ end of the PKD1 gene is at a CpG island which lies within the duplicated area. Homologous areas to this island, in the HG region, also have cleavable sites for methylation sensitive enzymes; these duplicate islands probably lie at the 5′ ends of the various HG genes. Analysis for DNAase hypersensitivity also indicates that the HG, CpG islands probably contain active promoters. These results are consistent with the observation of polyadenylated mRNA from the HG genes on Northern blots and the similarity of the expression pattern of the HG and PKD1 genes in different tissue specific cell lines. The HG genes may have complete open reading frames and may encode functional proteins. Antibodies to their ‘unique’ 3′ regions will be required to determine this. Although the PKD1 transcript is large, the overall size of the gene, at 52 kb, is not (the Duchenne muscular dystrophy (DMD) gene which encodes a slightly smaller transcript has a genomic size of over 2 Mb). Indeed, if the first intron of PKD1 is excluded from the analysis, 40.3% of the remainder of the gene is found in the mature mRNA. In the compact structure of the PKD1 gene, some of the introns are close to or smaller than the minimal size of 80 bp thought to be required for efficient splicing, although they are presumably excised effectively. We have shown that deletion of 18 or 10 bp from one small intron (intron 43), resulting in an intron of 55 or 57 bp, leads to aberrant splicing (Peral, 1995). Similar mutations may be found in the other small introns of this gene. The compact nature of the PKD1 gene probably reflects the GC rich area of the genome in which it is found (the PKD1 transcript has a total GC content of about 65%); a similar organisation is seen in other genes from the area of chromosome 16 (Vyas, 1992) is in an AT rich genomic region.

It is clear that polycystin has many features of a cell adhesion or recognition molecule with multiple different extracellular domains. These various binding domains are likely to have different specificities so that it can be envisaged that it will bind to a variety of different proteins (and carbohydrates) both on other cells and possibly in the extracellular matrix. Although provisional evidence indicates a wide range of expression of polycystin in tissue specific cell lines, detailed analysis by in situ of the mRNA and with antibodies to determine the cells expressing this protein both in adult tissue and during development will provide further evidence.

Initial analysis has revealed little clear evidence of alternate splicing, although one cDNA (out of 6 studied) had an extra exon of 255 bp positioned in intron 16. This exon contains an in-frame stop codon and it is not known at this stage if this represents an incompletely spliced mRNA or a splice form of polycystin which terminates at this point. Truncation of the protein here would leave a secreted protein lacking all of the transmembrane and cytoplasmic regions. Interestingly, a similar secreted form of the neural adhesion protein, NCAM, which is normally attached to the cell membrane, is produced by alternate splicing by insertion of an exon containing a stop codon (Gower et al., 1988).

The initial changes that have been noted in ADPKD kidneys are abnormal thickening and splitting of the basement membrane (BM) and simultaneous de-differentiation of associated epithelial cells at the point of tubular dilation. Similar results have been noted in the heterozygote Han:SPRD rat (Schafer et al., 1994) which is a dominant model of PKD, although it is not known if it is a rat model of PKD1. Concurrent changes in cellular characteristics and the BM suggests that a disruption or alteration of communication between the cell and the BM may be the primary change in this disease. Polycystin could play an important role in interaction and communication between epithelial cells and the BM. It is known that signals are required from cells to the extracellular matrix (ECM) for normal BM development and also that communication from the ECM to cells is required for control of cellular differentiation. Communication between the ECM and cells occurs by several different means including through integrins and so polycystin may bind to integrins, although it may interact directly with components of the ECM. Although ADPKD is generally a disease of adulthood, there is plenty of evidence that the cystic changes in the kidney may start much earlier (Milutinovic et al., 1970), even in utero (Reeders, 1986). Expression of polycystin during renal development may be when its major role occurs, perhaps in assembly of the BM and it is then that the errors, which later lead to cyst development, occur.

The plethora of connective tissue abnormalities associated with ADPKD indicate that the adhesion/communication roles of polycystin may be important for assembly and/or maintenance of the BM in many tissues, as well as the kidney. Hence, it is possible that disruption of normal cell adhesion and communication mediated by polycystin may explain the primary defects seen in the kidney and other organs in ADPKD. Clearly molecules that interact with polycystin or have a similar role are candidates for the other renal polycystic diseases of man.

A study of the mutations of the PKD1 gene highlight important functional regions of the protein. All of the mutations described so far in typical PKD1 families involve deletion or other disruption in the 3′ end of gene. Two large deletions detected on Southern blots remove a large part of the protein (or make an out of frame product) including the last 6 transmembrane domains and the C-terminal end. The in-frame splicing change described in the same paper would remove most of TM10 and part of the preceding cytoplasmic loop. Two recently described splicing mutations (Peral, 1995) create three different products which either delete part of the cytoplasmic loop between TM7 and TM8 or a larger region of this loop including part of TM7 or insert an extra region into that loop. These mutated genes may make functional protein (they all produce abnormal mRNA) and it is interesting to note that, in each case, these proteins would have an intact extracellular region with disrupted cytoplasmic and transmembrane areas. Such proteins may bind to extracellular targets but are unable to communicate in a normal way.

A group of mutations of PKD1 which completely delete the gene and hence are clearly inactivating have been described (Brook-Carter, 1994). However, in each of these cases the deletions also disrupt the adjacent TSC2 gene making interpretation of these cases difficult (TSC2 mutations alone can cause the development of renal cysts). Nevertheless, the severity of the polycystic disease in these patients indicate that inactivation of one PKD1 allele does promote cyst development. Further more, all these children are often severely affected at birth, cyst formation must occur in utero in these cases and hence polycystin has an important developmental role. A second somatic hit in the target tissue may also be required in these cases (and normal PKD1 patients) before cyst development can occur.

PKD1 Gene and Polycystic Kidney Disease

We have therefore compelling evidence that mutations of the PKD1 gene give rise to the typical phenotype of ADPKD. The location of this gene within the PKD1 candidate region and the available genetic evidence from the families with mutations show that this is the PKD1 gene. The present invention therefore includes the complete PKD1 gene itself and the six PKD1—associated mutations which have been described: a de novo translocation, which was subsequently transmitted with the phenotype; two intragenic deletions (one a de novo event); two further deletions; and a splicing defect.

It has been argued that PKD1 could be recessive at the cellular level, with a second somatic mutation required to give rise to cystic epithelium (Reeders, 1992). This “two hit” process is thought to be the mutational mechanism giving rise to several dominant diseases, such as neurofibromatosis (Legius, et al., 1993) and tuberous sclerosis (Green, et al., 1994) which result from a defect in the control of cellular growth. If this were the case, however, we might expect that a proportion of constitutional PKD1 mutations would be inactivating deletions as seen in these other disorders.

The location of the PKD1 mutations may, however, reflect some ascertainment bias as it is this single copy area which has been screened most intensively for mutations. Nevertheless, no additional deletions were detected when a large part of the gene was screened by FIGE, and studies by PFGE showed no large deletions of this area in 75 PKD1 patients. It is possible that the mutations detected so far result in the production of an abnormal protein which causes disease through a gain of function. However, it is also possible that these mutations eliminate the production of functional protein from this chromosome and result in the PKD1 phenotype by haploinsufficiency, or only after loss of the second PKD1 homologue by somatic mutation.

At least one mutation which seems to delete the entire PKD1 gene has been identified (WS-53) but in this case it also disrupts the adjacent TSC2 gene and the resulting phenotype is of TSC with severe cystic kidney disease. Renal cysts are common in TSC so that the phenotypic significance of deletion of the PKD1 gene in this case is difficult to assess. It is clear that not all cases of renal cystic disease in TSC are due to disruption of the PKD1 gene; chromosome 9 linked TSC (TSC1) families also manifest cystic kidneys and we have analysed many TSC2 patients with kidney cysts who do not have deletion of the PKD1 gene.

Preliminary analysis of the PKD1 protein sequence (SEQ ID NO. 8) has highlighted two regions which provide some clues to the possible function of the PKD1 gene. At the extreme 5′ end of the characterised region are two leucine-rich repeats (LRRs) (amino acids 29-74) flanked by characteristic amino flanking (amino acids 6-28) and carboxy flanking sequences (amino acids 76-133) (Rothberg et al., 1990). LRRs are thought to be involved in protein-protein interations (Kobe and Deisenhofer, 1994) and the flanking sequences are only found in extracellular proteins. Other proteins with LRRs flanked on the amino and carboxy sides are receptors or are involved in adhesion or cellular signalling. Further 3′ on the protein (amino acids 350-515) is a C-type lectin domain (Curtis et al., 1992). This indicates that this region binds carbohydrates and is also likely to be extracellular. These two regions of homology indicate that the 5′ part of the PKD1 protein is extracellular and involved in protein-protein interactions. It is possible that this protein is a constituent of, or plays a role in assembling, the extracellular matrix (ECM) and may act as an adhesive protein in the ECM. It is also possible that the extracellular portion of this protein is important in signalling to other cells. The function of much of the PKD1 protein is still not fully known but the presence of several hydrophobic regions indicates that the protein may be threaded through the cell membrane.

Familial studies indicate that de novo mutations probably account for only a small minority of all ADPKD cases; a recent study detected 5 possible new mutations in 209 families (Davies, et al., 1991). However in our study one of three intragenic muttions detected was a new mutation and the PKD1 associated translocation was also a de novo event. Furthermore, the mutations detected in the two familial cases do not account for a significant proportion of the local PKD1. The OX875 deletion was only detected in 1 of 282 unrelated cases, and the splicing defect was seen in only 1 of 48 unrelated cases. Nevertheless, studies of linkage disequilibrium have found evidence of common haplotypes associated with PKD1 in a proportion of some populations (Peral, et al., 1994; Snarey, et al., 1994) suggesting that common mutations will be identified.

Once a larger range of mutations have been characterised it will be possible to evaluate whether the type and location of mutation determines disease severity, and if there is a correlation between mutation and extra-renal manifestations. Previous studies have provided some evidence that the risk of cerebral aneurysms ‘runs true’ in families (Huston, et al., 1993) and that some PKD1 families exhibit a consistently mild phenotype (Ryynanen, et al., 1987). A recent study has concluded that there is evidence of anticipation in ADPKD families, especially if the disease is transmitted through the mother (Fink, et al., 1994). Furthermore, analysis of families with early manifestations of ADPKD show that there is a significant intra-familial recurrence risk and that childhood cases are most often transmitted maternally (Rink, et al., 1993; Zerres, et al., 1993). This pattern of inheritence is reminiscent of that seen in diseases in which an expanded trinucleotide repeat was found to be the mutational mechanism (reviewed in Mandel, 1993). However, no evidence for an expanding repeat correlating with PKD1 has been found in this region although such a sequence cannot be excluded.

There is ample evidence that early presymptomatic diagnosis of PKD1 is helpful because it allows complications such as hypertension and urinary tract infections to be monitored and treated quickly (Ravine, et al., 1991). The identification of mutations within a family allow rapid screening of that and other families with the same mutation. However, genetic linkage analysis is likely to remain important for presymptomatic diagnosis. The accuracy and ease of linkage based diagnosis will be improved by the identification of the PKD1 gene as a microsatellite lies in the 3′ untranslated region of this gene (KG-8) and several CA repeats are located 5′ of the gene (see FIGS. 1a and 6; Peral, et al., 1994; Snarey, et al., 1994).

Experimental Procedures

Clinical Details of Patients

Family 77

77-2 and 77-3 are 48 and 17 years old, respectively and have typical ADPKD. Both have bilateral polycystic kidneys and 77-2 has impaired renal function. Neither patient manifests any signs of TSC (apart from cystic kidneys) on clinical and ophthalmological examination or by CT scan of the brain.

77-4 is 13 years old, severely mentally retarded and has multiple signs of tSC including adenoma sebaceum, depigmented macules and periventricular calcification on CT scan. Renal ultrasound reveals a small number of bilateral renal cysts.

ADPKD Patients

OX875 developed ESRD from ADPKD, aged 46. Progressive decline in renal function had been observed over 17 years; ultrasound examinations documented enlarging polycystic kidneys with less extensive hepatic cystic disease. Both kidneys were removed after renal transplantation and pathological examination showed typical advanced cystic disease in kidneys weighing 1920 g and 340 g (normal average 120 g).

OX114 developed ESRD from ADPKD aged 54: diagnosis was made by radiological investigation during an episode of abdominal pain aged 25. A progressive decline in renal function and the development of hypertension was subsequently observed. Ultrasonic examination demonstrated enlarged kidneys with typical cystic disease, with less severe hepatic involvement.

OX32 is a member of a large kindred affected by typical ADPKD in which several members have developed ESRD. The patient himself has been observed for 12 years with progressive renal failure and hypertension following ultrasonic demonstration of polycystic kidneys.

No signs of TSC were observed on clinical examination of any of the ADPKD patients.

DNA Electrophoresis and Hybridisation

DNA extraction, restriction digests, electrophoresis, Southern blotting, hybridisation and washing were performed by standard methods or as previously described (Harris, et al., 1990). FIGE was performed with the Biorad FIGE Mapper using programme 5 to separate fragments from 25-50 kb. High molecular weight DNA for PFGE was isolated in agarose blocks and separated on the Biorad CHEF DRII apparatus using appropriate conditions.

Genomic DNA Probes and Somatic Cell Hybrids

Many of the DNA probes used in this study have been described previously: MS205.2 (D16S309; Royle, et al., 1992); GGG1 (D16S259; Germino, et al., 1990); N54 (D16S139; Himmelbauer, et al., 1991); SM6 (D16S665), CW23, CW21, and JH1 (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). Microsatellite probes for haplotype analysis were KG8 and W5.2 (Snarey, et al., 1994) SM6, CW3 and CW2, (Peral, et al., 1994), 16AC2.5 (Thompson, et al., 1992); SM7 (Harris, et al., 1991), VK5AC (Aksentijevich, et al., 1993).

New probes isolated during this study were: JH4, JH5, JH6, 11 kb, 6 kb and 6 kb BamH I fragments, respectively, and JH13 and JH14, 4 kb and 2.8 kb BamH I-EcoR I fragments, respectively, all from the cosmid JH2A; JH8 and JH10 are 4.5 kb and 2 kb Sac I fragments, respectively and JH12 a 0.6 Sac I-BamH I fragment, all from JH4; 8S1 and 8S3 are 2.4 kb and 0.6 kb Sac II fragments, respectively, from JH8; CW10 (SEQ ID NO. 4) is a 0.5 kb Not I-Mlu I fragment of SM25A; JH17 is a 2 kb EcoR I fragment of NM17.

The somatic cell hybrids N-OH1 (Germino, et al., 1990), P-MWH2A (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and Hy145.19 (Himmelbauer, et al., 1991) have previously been described. Somatic cell hybrids containing the paternally derived (BP2-10) and maternally derived (BP2-9) chromosomes from OX114 were produced by the method of Deisseroth and Hendrick (1979).

Constructing a Cosmid Contig

Cosmids were isolated from chromosome 16 specific and total genomic libraries, and a contig was constructed using the methods and libraries previously described (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). To ensure that cosmids were derived from the 16p13.3 region (not the duplicate 16p13.1 area) initially, probes from the single copy area were used to screen libraries (e.g. CW21 and N54). Two cosmids mapped entirely within the area duplicated, CW10III and JC10.2B. To establish that these were from the PKD1 area, they were restriction mapped and hybridised with the probe CW10. The fragment sizes detected were compared to results obtained with hybrids containing only the 16p13.3. are (Hy145.19) or only the 16p13.1 region (P-MWH2A).

FISH

FISH was performed essentially as previously described (Buckle and Rack, 1993). The hybridisation mixture contained 100 ng of biotin-II-dUTP labelled cosmid DNA and 2.5 mg human Cot-1 DNA (BRL), which was denatured and annealled at 37° C. for 15 min prior to hybridisation at 42° C. overnight. After stringent washes the site of hybridisation was detected with successive layers of fluorescein-conjugated avidin (5 mg/ml) and biotinylated ani-avidin (5 mg/ML) Vector Laboratories). Slides were mounted in Vectashield (Vector Laboratories) containing 1 mg/ml propidium iodide and 1 mg/ml 4′, 6-diamidino-2-phenylindole (DAPI), to allow concurrent G-banded analysis under UV light. Results were analysed and images captured using a Bio-Rad MRC 600 confocal laser scanning microscope.

cDNA Screening and Characterisation

Foetal brain cDNAs libraries in 7 phage (Clonetech and Stratagene) were screened by standard methods with genomic fragments in the single copy area (equivalent to CW23 and CW21) or with a 0.8 kb Pvu II-Eco RI single copy fragment of AH3. Six PBP cDNAs were characterised; AH4 (1.7 kb) and 3A3 (2.0 kb) are described in European Chromosome 16 Tuberous Sclerosis Consortium, 1993, and four novel cDNAs AH3 (2.2 kb), AH6(2.0 kb), A1C (2.2 kb) and B1E (2.9 kb). A Striatum library (Stratagene) was screened with JH4 and a HG-C cDNA, 11BHS21 (3.8 KB) WAS ISOLATED, 21p.9 is a 0.9 kb Pvu II-EcoR I subclone of this cDNA. A HG-A or HG-B cDNA, HG-4 (7 kb) was also isolated by screening the foetal brain library (Stratagene) with JH8. HG-4/1.1 is a 1.1 kb Pvu II-EcoR I fragment from the 3′ end of HG-4. 1A1H.6 (SEQ ID NO. 3) is a 0.6 kb Hind III-EcoR I subclone of a TSC2 CDNA, 1A-1 (1.7 kb), which was isolated from the Clonetech library. Each cDNA was subcloned into Bluescript and sequenced utilising a combination of sequential truncation and liigonucleotide primers using DyeDeoxy Terminators (Applied Biosystems) and an ABI 373A DNA Sequencer (Applied Biosystems) or by hand with ‘Sequenase’ T7 DNA polymerase OUSB).

RNA Procedures

Total RNA was isolated from cell lines and tissues by the method of Chomczynskiand Sacchi (1987) and enrichment for mRNA made using the PolyAT tract mRNA Isolation System (Promega). For RNA electrophoresis 0.5% agarose denaturing formaldehyde gels were used which were Northern blotted, hybridised and washed by standard procedures. The 0.24-9.5 kb RNA (Gibco BRL) size standard was used and hybridisation of the probe (1-9B3) to the 13 kb Utrophin transcript (Love, et al., 1989) in total fibroblast RNA was used as a size marker for the large transcripts.

RT-PCR was performed with 2.5 mg of total RNA by the method of Brown et al. (1990) with random hexamer primers, except that AMV-reverse transcriptase (Life Sciences) was employed. To characterise the deletion of the PBP transcript in OX114 we used the primers:

 AH# F9 5′ TTT GAC AAG CAC ATC TGG CTC TC 3′  (SEQ ID NO.9)

AH3 B7 5′ TAC ACC AGG AGG CTC CGC AG 3′  (SEQ ID NO.10)

in a DMSO containing PCR buffer (Dode, et al., 1990) with 0.5 mM MgCl₂ and 36 cycles of: 94° C., 1 min; 61° C., 1 min; 72° C., 2 min plus a final extension of 10 min. The 3A3 C primers used to amplify the OX32 CDNA and DNA were:

3A3 C1 5′ CGC CGC TTC ACT AGC TTC GAC 3′  (SEQ ID NO. 11)

3A3 C2 5′ ACG CTC CAG AGG GAG TCC AC 3′  (SEQ ID NO. 12)

These were employed in a PCR buffer and cycle previously described (Harris, et al., 1991) with 1 mM MgCl₂ and an annealing temperature of 61° C.

PCR products for sequencing were amplified with Pfu-1 (Stratagene) and ligated into the Srf-1 site in PCR-Script (Stratagene) in the presence of Srf-1.

RNAse Protection

Tissues from normal and end-stage polycystic kidneys were immediately homogenised in guanidinium thiocyanate. RNA was purified on a cesium chloride gradient and 30 mg total RNA was assayed by RNAse protection by the method of Melton, et al., (1984) using a genomic template generated with the 3A3, C primers (SEQ ID NOS. 11 and 12).

Heteroduplex Analysis

Heteroduplex analysis was performed essentially as described by Keen et al. (1991). Samples were amplified from genomic DNA with the 3A3, C primers (SEQ ID NOS. 11 and 12), heated at 95° C. for 5 minutes and incubated at room temperature for at least 30 minutes before loading on a Hydrolink gel (AT Biochem). Hydrolink gels were run for 12-18 hours at 250V and fragments observed after staining with ethidium bromide.

Extraction and Amplification of Paraffin-embedded DNA

DNA from formalin fixed, paraffin wax embedded kidney tissue was prepared by the method of Wright and Manos (1990), except that after proteinase K digestion overnight at 55° C., the DNA was extracted with phenol plus chloroform before ethanol precipitation. Approximately 50 ng of DNA was used for PCR with 1.5 mM MgCl₂ and 40 cycles of 94° C. for 1 min, 50° C. for 1 min and 72° C. for 40 s, plus a 10 min extension at 72° C.

The oligonucleotide primers designed to amplify across the genomic deletion of OX875 were:

AHF42: 5′-GGG CAA GGG AGG ATG ACA AG-3′  (SEQ ID NO. 13)

JH14B3 : 5′-GGG TTT ATC AGC AGC AAG CGG-3′

which produced a product of about 220 bp in individuals with the OX875 deletion.

3′ RACE Analysis of WS-212

3′ RACE was completed essentially as described (European Polycystic Kidney Disease Consortium (1994)). Reverse transcription was performed with 5 μg total RNA with 0.5 μg of the hybrid dT₇ adapter primer using conditions previously described (Fronman et al., (1988)). A specific 3′ RACE product was amplified with the primer F5 and adapter primer in 0.5 mM MgCl₂ with the program: 57° C., 60 s; 72° C., 15 minutes and 30 cycles of 95° C., 40 s; 57° C., 60 s; 72° C., 60 s plus 72° C., 10 minutes. The amplified product was cloned using the TA cloning system (Invitrogen) and sequenced by conventional methods.

Genomic and cDNA Probes and Somatic Cell Hybrids

The genomic clones CW21, JH5, JH6, JH8, JH10, JH12, JH13 and JH14 and the cDNAs A1C, AH3, 3A3 and AH4 are described herein. Newly described probes are: SM3 a 2.0 kb BamH l subclone of the cosmid SMll, JH9, 2.4 kb Sac l fragment and JH11, 1.2 kn Sac l—BamHl fragment, both from JH4. See Eur. Polycystic Kidney Disease. Consortium, 1994 and Eur. Chromosome 16 Tuberous sclerosis Consortium 1993 for all above clones. DFS5 is a 4.2 kb Not l-Hind lll fragment of CW23 (Eur. Chromosome 16 Tuberous Sclerosis Consortium, 1993). The cDNAs; BPG4, BPG6, BPG7C and 13-A were isolated from a fetal brain cDNA library in X phage (Stratagene) and are 7 kb, 2 kb, 4.5 kb and 1.2 kb respectively.

The somatic cell hybrids have previously been described, P-MWH2A (Eur. Chromosome 16 Tuberous Sclerosis Consortium, 1993) and Hy145.19 (Himmelbauer et al., 1991).

Exon Linking

Total cellular RNA from the radiation hybrid Hy145.19 was reverse transcribed using random hexamers (Eur. Polycystic Kidney Disease Consortium, 1994). This material was used as a template for PCR using the proof reading polymerase Pfu-1 with the primer pairs described in Table 2 (SEQ ID NO.7). The resultant products were cloned into the Srf-1 site of pPCRscript (SK+) plasmid.

Sequencing

Full length sequence was obtained from the genomic clones, HG cDNAs and exon link clones using the progressive unidirectional deletion technique of Henikoff, (1984). Both strands were then sequenced using DyeDeoxy Terminator Cycle Sequencing and an Applied Biosystems Sequencer 373A. Contig assembly was done using the programmes Assembly line (vs 1.0.7), SeqEd (vs 1.03) and MacVector (4.1.4).

Primer Extension

Primer extension was performed on total cellular fibroblast RNA. 25 μg of RNA was annealed at 60° C. in the presence of 400 mM NaCl to 0.01 pM of HPLC pure oligonucleotide which had been end labelled to a specific activity of 3×10⁷ cpm/pM with ³²P. Primer extension was then performed in the presence of 50 mM Tris pH8.2, 10 mM DTT, 6 mM MgCl₂, 25 mg/ml Actinomycin D, 0.5 mM dNTPs, and 8 units of AMV reverse transcriptase. The extension reaction was continued for 60 min at 42° C. The extension products were compared to a sequencing ladder generated using the same primer on the genomic clone SM3. The primers used were:

N2765:5′-GGCGCGGCGGGCGGCATCGTTAGGGCAGCG-3′  (SEQ ID NO.15)

N5496:5′-GGCGGGCGGCATCGTTAGGGCAGCGCGCGC-3′  (SEQ ID NO.16)

N5495:5′-ACCTGCTGCTGAGCGACGCCCGCTCGGGGC-3′  (SEQ ID NO.17)

Analysis of Sequence Homology

The predicted PKD1 protein was analyzed for homologies with known proteins in the SwissProt and NBRF database using the BLAST (Altschul et al., 1990) and FASTA (Pearson et al., 1988) algorithms. Layouts were prepared by hand and using the programme Pileup.

Transmembrane Regions

Potential transmembrane segments were identified by the method of Sipos and von Heljne (Sipos et al., 1993), using the GES hydrophobicity scale (Engelmen et al., 1986) and a trapezoid sliding window (a full window of 21 residues and a core window of 11 residues), as recommended. Candidate transmembrane domains were selected on the basis of their average hydrophobicity <H>, and were classified as certain (<H>≧1.0) or putative (0.6, <H><1).

The best topology for the protein was predicted on the basis of three different criteria: a) the net charge difference between the 15 N-terminal and the 15 C-terminal residues flanking the most N-terminal transmembrane segment (Hartmann et al., 1989); b) the difference in positively charged residues between the two sides of the membrane in loops smaller than 60 residues, and c) the analysis of the overall amino acid composition of loops longer than 60 residues by the compositional distance method (Nakashima et al., 1992). Using the above criteria the TopPred II program (Sipos wt al., 1993) calculated all the possible topologies of the proteins including the certain transmembrane segments and either included or excluded each of the putative segments to determine the most likely structure.

PKD1 Protein Purification

The PKD1 protein may be purified according to conventional protein purification procedures well known in the art. Alternatively, the protein may be purified from cells harboring a plasmid containing an expressible PKD1 gene. For example, the protein may be expressed in an E.coli expression system and purified as follows.

Cells are grown in a 10 liter volume in a Chemap Fermentor (Chemapec, Woodbury, N.Y.) in 2% medium. Fermentation temperature may be 37° C., pH 6.8, and air as provided at 1 vvm. Plasmid selection may be provided using ampicillin for a plasmid containing an ampicillin resistance gene. Typical yield (wet weight) is 30 g/l.

For cell lysis, 50 g wet cell weight of E.coli containing the recombinant PKD1 plasmid may be resuspended in a final volume of 100 ml in 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 5 mM DTT, 15 mM mercaptoethanol, 0.5% triton X-100, and 5 mM PMSF. 300 mg lysozyme is added to the suspension, and incubated for 30 min at room temperature. The material is then lyzed using a BEAD BEATER (R) (Biospec Products, Bartlesville, Okla.) containing an equal volume of 0.1-0.15 um glass beads. The liquid is separated from the beads and the supernatant removed, the pellet dissolved in 20 mM Tris-Cl pH 8.0.

The protein may. be purified from the supernatant using DEAE chromatography, as is well known in the art.

Preparation of Antibodies

Antibodies specific for PDK1 protein or a fragment thereof are prepared as follows. A peptide corresponding to at least 8 amino acid residues of the PKD1 sequence of FIG. 15 (SEQ ID NO.8), are synthesized. Coupling of the peptide to carrier protein and immunizations is performed as described (Dymecki, S. M., J. Biol. Chem 267:4815-4823, 1992). Rabbit antibodies against this peptide are raised and sera are titered against peptide antigen by ELISA. The sera exhibiting the highest titer (1:27,000) are most useful.

Techniques for preparing monoclonal antibodies are well known, and monoclonal antibodies of this invention may be prepared by using the synthetic: polypeptides of this invention, preferably bound to a carrier, as the immunogen as was done by Arnheiter et al., Nature, 294, 278-280 (1981).

Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced. Nevertheless, monoclonal antibodies may be described as being “raised to” or “induced by” the synthetic polypeptides of this invention or their conjugates with a carrier.

Antibodies are utilized along with an “indicating group” also sometimes referred to as a “label”. The indicating group or label is utilized in conjunction with the antibody as a means for determining whether an immune reaction has taken place, and in some instances for determining the extent of such a reaction.

The indicating group may be a single atom as in the case of radioactive elements such as iodine 125 or 131, hydrogen 3 or sulfur 35, or NMR-active elements such as fluorine 19 or nitrogen 15. The indicating group may also be a molecule such as a fluorescent dye like fluorescein, or an enzyme, such as horseradish peroxidase (HRP), or the like.

The terms “indicating group” or “label” are used herein to include single atoms and molecules that are linked to the antibody or used separately, and whether those atoms or molecules are used alone or in conjunction with additional reagents. Such indicating groups or labels are themselves well-known in immunochemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel antibodies, methods and/or systems.

Detection of PKD1 and Subcellular Localization

Another embodiment of this invention relates to an assay for the presence of PKD1 protein in cells. Here, an above-described antibody is raised and harvested. The antibody or idiotype-containing polyamide portion thereof is then admixed with candidate tissue and an indicating group. The presence of the naturally occurring amino acid sequence is ascertained by the formation of an immune reaction as signaled by the indicating group. Candidate tissues include any tissue or cell line or bodily fluid to be tested for the presence of PKD1.

Metabolic labeling immunoprecipitation, and immunolocalization assays are performed in cells as described previously (Furth, M. E., et al., Oncogene 1:47-58, 1987; Laemmli, U. K., Nature 227:680-685, 1970; Yarden, Y., et al., EMBO J. 6:3341-3351, 1987; Konopka, J. B., et al., Mol. Cell. Biol. 5:3116-3123, 1985). For immunoblot analysis, total lysates are prepared (using Fruth's lysis buffer) (Fruth, M. E., et al., Oncogene, 1:47-58, 1987). Relative protein concentrations are determined with a colorimetric assay kit (Bio-Rad) with bovine serum albumin as the standard. A protein of lysate containing approximately 0.05 mg of protein is mixed with an equal volume of 2×SDS sample buffer containing 2 mercaptoethanol, boiled for 5 min., fractioned on 10% polyacrylamide-SDS gels (Konopka, J. B., et al., J.Virol., 51:223-232, 1984) and transferred to immunobilon polyvinyldine difluoride (Millipore Corp., Bedford, Mass.) filters. Protein blots are treated with specific antipeptide antibodies (see below). Primary binding of the PKD1-specific antibodies is detected using anti-IgG second antibodies conjugated to horseradish peroxidase and subsequent chemiluminescence development ECL Western blotting system (Amersham International).

For metabolic labeling, 10⁶ cells are labeled with 100 μCi of ³⁵S-methionine in 1 ml of Dulbecco's modified Eagles medium minus methionine (Amersham Corp.) for 16 h. Immunoprecipitation of PKD1 protein from labeled cells with antipeptide antiserum is performed as described (Dymecki, S. M., et al., supra). Portions of lysates containing 10⁷ cpm of acid-insoluble ³⁵S-methionine are incubated with 1 μg of the antiserum in 0.5 ml of reaction mixture. Immunoprecipitation samples are analyzed by SDS-polylarcylamide gel electrophoresis and autoradiography.

For immunolocalization studies, 10⁷ CMK cells are resuspended in 1 ml of sonication buffer (60 mM Tris-HCl, pH 7.5, 6mM EDTA, 15 mM EGTA, 0.75M sucrose, 0.03% leupeptin 12 mM phenylmethylsulfonyl fluoride, 30 mM 2-mercaptoethanol). Cells are sonicated 6 times for 10 seconds each and centrifuged at 25,000×g for 10 min at 4° C. The pellet is dissolved in 1 ml of sonication buffer and centrifuged at 25,000×g for 10 min at 4° C.

The pellet (nucleus fraction) is resuspended in 1 ml of sonication buffer and added to an equal volume of 2×SDS sample buffer. The supernatant obtained above (after the first sonication) is again centrifuged at 100,000×g for 40 min at 4° C. The supernatant (cytosolic fraction) is removed and added to an equal volume of 2×concentrated SDS sample buffer. The remaining pellet (membrane fraction) is washed and dissolved in sonication buffer and SDS sample buffer as described above. Protein samples are analyzed by electrophoresis on 10% polyacrylamide gels, according to the Laemmli method (Konopka, J. B., supra) The proteins are transferred from the gels on a 0.45-μm polyvinylidine difluoride membrane for subsequent immunoblot analysis. Primary binding of the PKD1 specific antibodies is detected using anti-IgG second antibodies conjugated to horseradish peroxidase.

For immunohistochemical localization of PKD1 protein, CMK cells or U3T3 are grown on cover slips to approximately 50% confluence and are washed with PBS (pH 7.4) after removing the medium. The cells are prefixed for 1 min at 37° C. in 1% paraformaldehyde containing 0.075% Triton X-100, rinsed with PBS and then fixed for 10 min with 4% paraformaldehyde. After the fixation step, cells are rinsed in PBS, quenched in PBS with o.1 and finally rinsed again in PBS. For antibody staining, the cells are first blocked with a blocking solution (3% bovine serum albumin in PBS) and incubated for 1 h at 37° C. The cells are then incubated for 1 h at 37° C. with antiserum (1:100 dilution or with preimmune rabbit serum (1:100). After the incubation with the primary antibody, the cells are washed in PBS containing 3% bovine and serum albumin and 0.1% Tween 20 and incubated for 1 h at 37° C. in fluorescein-conjugated donkey anti-rabbit IgGs (Jackson Immunoresearch, Maine) diluted 1:100 in blocking solution.

The coverslips are washed in PBS (pH 8.0), and glycerol is added to each coverslip before mounting on glass slides and sealing with clear nail polish. All glass slides are examined with a Zeiss Axiophot microscope.

An indicating group or label is preferably supplied along with the antibody and may be packaged therewith or packaged separately. Additional reagents such as hydrogen peroxide and diaminobenzideine may also be included in the system when an indicating group such as HRP is utilized. Such materials are readily available in commerce, as are many indicating groups, and need not be supplied along with the diagnostic system. In addition, some reagents such as hydrogen peroxide decompose on standing, or are otherwise short-lived like some radioactive elements, and are better supplied by the end-user.

Pharmaceutical Compositions of the Invention; Dosage and Administration

Pharmaceutical formulations comprising PKD1 nucleic acid or protein, or mutants thereof, can be prepared by procedures well known in the art. For example, as injectables, e.g., liquid solutions or suspensions. Solid forms for solution in, or suspension in, a liquid prior to injection also can be prepared. Optionally, the preparation also can be emulsified. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. For example, water, saline, dextrose, glycerol, ethanol, etc. or combinations thereof. Also useful are wetting or emulsifying agents, pH buffering agents or adjuvants. PKD1 protein or DNA can be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. In each case, the active protein or the nucleic acid will be present in the range of about 0.05% to about 10%, preferably in the range of about 1-2% by weight. Alternatively, the active protein or the nucleic acid will be administered at a dosage of about 10 mg-2 kg/kg body weight, preferably 50 mg-400 mg/kg/body weight. Administration may be daily, weekly, or in a single dosage, as determined by the physician.

TABLE 1 Details of the exons and introns of the PKD1 gene Exons Introns Exon position Size (nt) positions Intron Size No. (bp) bp (aa) No. (bp) 1  1-426 426  1-72 1 ˜17 kb 2 427-498 72 73-96 2 121 3 499-570 72  97-120 3 268 4 571-740 170 121-177 4 213 5  741-1412 672 177-401 5 117 6 1413-1596 184 401-462 6 435 7 1597-1817 221 463-536 7 188 8 1818-1933 118 536-575 8 410 9 1934-2060 127 525-617 9 363 10 2061-2308 248 617-700 10 452 11 2309-3064 756 700-952 11 877 12 3065-3196 132 952-996 12 196 13 3197-3372 176  996-1054 13 314 14 3373-3506 134 1055-1099 14 468 15 3507-7126 3,620 1099-2306 15 219 16 7127-7276 150 2306-2356 16 ? 17 7277-7420 144 2356-2404 17 127 18 7421-7700 280 2404-2497 18 93 19 7701-7914 214 2497-2568 19 66 20 7915-8074 160 2569-2622 20 ˜400 bp 21 8075-8227 153 2622-2673 21 3.1 kb 22 8228-8372 145 2673-2721 22 650 23 8373-9002 630 2721-2931 23 295 24 9003-9159 158 2931-2983 24 180 25 9160-9412 254 2984-3068 25 123 26 9413-9608 196 3068-3133 26 ˜1.7 kb 27 9609-9779 171 3133-3190 27 86 28 9780-9923 144 3190-3238 28 93 29  9924-10134 211 3238-3308 29 90 30 10135-10261 127 3309-3351 30 ˜1.8 kb 31 10262-10378 117 3351-3390 31 88 32 10379-10428 50 3390-3406 32 224 33 10429-10613 185 3407-3468 33 77 34 10614-10707 94 3468-3499 34 ˜3 kb 35 10708-10826 119 3500-3539 35 78 36 10827-11029 203 3539-3607 36 72 37 11030-11224 195 3607-3672 37 450 38 11225-11364 140 3672-3718 38 361 39 11365-11477 113 3719-3756 39 290 40 11478-11619 142 3756-3803 40 139 41 11620-11745 126 3804-3845 41 183 42 11746-11920 175 3846-3904 42 ˜320 43 11921-12211 291 3904-4001 43 75 44 12212-12346 135 4001-4046 44 83 45 12347-12652 306 4046-4148 45 88 46 12653-14148 1,496 4148-4302

TABLE 2 Details of the exon link cDNAs Product Product Position Exon Name Size (bp) Oligonucleotide Sequences in cDNA Position rev1 652 AGCGCCAGCGTCCGAGCGG  8-658 1-4 CTGCACCACCCGCACCTGC 200-658 S13 1285 CCGGGCGCTGGACGTTGGGCT 448-1733 2-7 AGTGCTCGGCTGTGGCTGGGT S3/4 1608 CACCCAGCCACAGCCGAGCACT 1712-3320  7-13 GTGTGGCATTGGGGGACAGCAC S1/3 732 TGCTGTCCCCCAATGCCAC 3300-4032 13-15 ACGGTCACTGTGCAGTTC GAP e 1983 CCAATGCCACACTGGTACTGACG 3309-5292 13-15 TGGTAGGTGCCGGCCTCGAG GAP d 2036 CCGGCACCTACCATGTGCAGC 5280-7316 15-17 CCAAGGACACAATGGGCACC GAP g 884 GAGGTGTATCGCACCGCCAG 6773-7657 15-18 GCCCAGTGGGAAGAGGCGGC GAP a 1211 TCTTGCCGCCTCTTCCCA 7634-8862 18-23 GCAGCCCAGTCCGAGTTG

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited thereto, being defined by the claims set forth below.

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23 5631 base pairs nucleic acid unknown unknown cDNA Homo sapiens CDS 1..4842 misc_feature 1..5631 /function= “Original 3′ end of the PKD1 gene” 1 CTC AAC GAG GAG CCC CTG ACG CTG GCG GGC GAG GAG ATC GTG GCC CAG 48 Leu Asn Glu Glu Pro Leu Thr Leu Ala Gly Glu Glu Ile Val Ala Gln 1 5 10 15 GGC AAG CGC TCG GAC CCG CGG AGC CTG CTG TGC TAT GGC GGC GCC CCA 96 Gly Lys Arg Ser Asp Pro Arg Ser Leu Leu Cys Tyr Gly Gly Ala Pro 20 25 30 GGG CCT GGC TGC CAC TTC TCC ATC CCC GAG GCT TTC AGC GGG GCC CTG 144 Gly Pro Gly Cys His Phe Ser Ile Pro Glu Ala Phe Ser Gly Ala Leu 35 40 45 GCC AAC CTC AGT GAC GTG GTG CAG CTC ATC TTT CTG GTG GAC TCC AAT 192 Ala Asn Leu Ser Asp Val Val Gln Leu Ile Phe Leu Val Asp Ser Asn 50 55 60 CCC TTT CCC TTT GGC TAT ATC AGC AAC TAC ACC GTC TCC ACC AAG GTG 240 Pro Phe Pro Phe Gly Tyr Ile Ser Asn Tyr Thr Val Ser Thr Lys Val 65 70 75 80 GCC TCG ATG GCA TTC CAG ACA CAG GCC GGC GCC CAG ATC CCC ATC GAG 288 Ala Ser Met Ala Phe Gln Thr Gln Ala Gly Ala Gln Ile Pro Ile Glu 85 90 95 CGG CTG GCC TCA GAG CGC GCC ATC ACC GTG AAG GTG CCC AAC AAC TCG 336 Arg Leu Ala Ser Glu Arg Ala Ile Thr Val Lys Val Pro Asn Asn Ser 100 105 110 GAC TGG GCT GCC CGG GGC CAC CGC AGC TCC GCC AAC TCC GCC AAC TCC 384 Asp Trp Ala Ala Arg Gly His Arg Ser Ser Ala Asn Ser Ala Asn Ser 115 120 125 GTT GTG GTC CAG CCC CAG GCC TCC GTC GGT GCT GTG GTC ACC CTG GAC 432 Val Val Val Gln Pro Gln Ala Ser Val Gly Ala Val Val Thr Leu Asp 130 135 140 AGC AGC AAC CCT GCG GCC GGG CTG CAT CTG CAG CTC AAC TAT ACG CTG 480 Ser Ser Asn Pro Ala Ala Gly Leu His Leu Gln Leu Asn Tyr Thr Leu 145 150 155 160 CTG GAC GGC CAC TAC CTG TCT GAG GAA CCT GAG CCC TAC CTG GCA GTC 528 Leu Asp Gly His Tyr Leu Ser Glu Glu Pro Glu Pro Tyr Leu Ala Val 165 170 175 TAC CTA CAC TCG GAG CCC CGG CCC AAT GAG CAC AAC TGC TCG GCT AGC 576 Tyr Leu His Ser Glu Pro Arg Pro Asn Glu His Asn Cys Ser Ala Ser 180 185 190 AGG AGG ATC CGC CCA GAG TCA CTC CAG GGT GCT GAC CAC CGG CCC TAC 624 Arg Arg Ile Arg Pro Glu Ser Leu Gln Gly Ala Asp His Arg Pro Tyr 195 200 205 ACC TTC TTC ATT TCC CCG GGG AGC AGA GAC CCA GCG GGG AGT TAC CAT 672 Thr Phe Phe Ile Ser Pro Gly Ser Arg Asp Pro Ala Gly Ser Tyr His 210 215 220 CTG AAC CTC TCC AGC CAC TTC CGC TGG TCG GCG CTG CAG GTG TCC GTG 720 Leu Asn Leu Ser Ser His Phe Arg Trp Ser Ala Leu Gln Val Ser Val 225 230 235 240 GGC CTG TAC ACG TCC CTG TGC CAG TAC TTC AGC GAG GAG GAC ATG GTG 768 Gly Leu Tyr Thr Ser Leu Cys Gln Tyr Phe Ser Glu Glu Asp Met Val 245 250 255 TGG CGG ACA GAG GGG CTG CTG CCC CTG GAG GAG ACC TCG CCC CGC CAG 816 Trp Arg Thr Glu Gly Leu Leu Pro Leu Glu Glu Thr Ser Pro Arg Gln 260 265 270 GCC GTC TGC CTC ACC CGC CAC CTC ACC GCC TTC GGC GCC AGC CTC TTC 864 Ala Val Cys Leu Thr Arg His Leu Thr Ala Phe Gly Ala Ser Leu Phe 275 280 285 GTG CCC CCA AGC CAT GTC CGC TTT GTG TTT CCT GAG CCG ACA GCG GAT 912 Val Pro Pro Ser His Val Arg Phe Val Phe Pro Glu Pro Thr Ala Asp 290 295 300 GTA AAC TAC ATC GTC ATG CTG ACA TGT GCT GTG TGC CTG GTG ACC TAC 960 Val Asn Tyr Ile Val Met Leu Thr Cys Ala Val Cys Leu Val Thr Tyr 305 310 315 320 ATG GTC ATG GCC GCC ATC CTG CAC AAG CTG GAC CAG TTG GAT GCC AGC 1008 Met Val Met Ala Ala Ile Leu His Lys Leu Asp Gln Leu Asp Ala Ser 325 330 335 CGG GGC CGC GCC ATC CCT TTC TGT GGG CAG CGG GGC CGC TTC AAG TAC 1056 Arg Gly Arg Ala Ile Pro Phe Cys Gly Gln Arg Gly Arg Phe Lys Tyr 340 345 350 GAG ATC CTC GTC AAG ACA GGC TGG GGC CGG GGC TCA GGT ACC ACG GCC 1104 Glu Ile Leu Val Lys Thr Gly Trp Gly Arg Gly Ser Gly Thr Thr Ala 355 360 365 CAC GTG GGC ATC ATG CTG TAT GGG GTG GAC AGC CGG AGC GGC CAC CGG 1152 His Val Gly Ile Met Leu Tyr Gly Val Asp Ser Arg Ser Gly His Arg 370 375 380 CAC CTG GAC GGC GAC AGA GCC TTC CAC CGC AAC AGC CTG GAC ATC TTC 1200 His Leu Asp Gly Asp Arg Ala Phe His Arg Asn Ser Leu Asp Ile Phe 385 390 395 400 CGG ATC GCC ACC CCG CAC AGC CTG GGT AGC GTG TGG AAG ATC CGA GTG 1248 Arg Ile Ala Thr Pro His Ser Leu Gly Ser Val Trp Lys Ile Arg Val 405 410 415 TGG CAC GAC AAC AAA GGG CTC AGC CCT GCC TGG TTC CTG CAG CAC GTC 1296 Trp His Asp Asn Lys Gly Leu Ser Pro Ala Trp Phe Leu Gln His Val 420 425 430 ATC GTC AGG GAC CTG CAG ACG GCA CGC AGC GCC TTC TTC CTG GTC AAT 1344 Ile Val Arg Asp Leu Gln Thr Ala Arg Ser Ala Phe Phe Leu Val Asn 435 440 445 GAC TGG CTT TCG GTG GAG ACG GAG GCC AAC GGG GGC CTG GTG GAG AAG 1392 Asp Trp Leu Ser Val Glu Thr Glu Ala Asn Gly Gly Leu Val Glu Lys 450 455 460 GAG GTG CTG GCC GCG AGC GAC GCA GCC CTT TTG CGC TTC CGG CGC CTG 1440 Glu Val Leu Ala Ala Ser Asp Ala Ala Leu Leu Arg Phe Arg Arg Leu 465 470 475 480 CTG GTG GCT GAG CTG CAG CGT GGC TTC TTT GAC AAG CAC ATC TGG CTC 1488 Leu Val Ala Glu Leu Gln Arg Gly Phe Phe Asp Lys His Ile Trp Leu 485 490 495 TCC ATA TGG GAC CGG CCG CCT CGT AGC CGT TTC ACT CGC ATC CAG AGG 1536 Ser Ile Trp Asp Arg Pro Pro Arg Ser Arg Phe Thr Arg Ile Gln Arg 500 505 510 GCC ACC TGC TGC GTT CTC CTC ATC TGC CTC TTC CTG GGC GCC AAC GCC 1584 Ala Thr Cys Cys Val Leu Leu Ile Cys Leu Phe Leu Gly Ala Asn Ala 515 520 525 GTG TGG TAC GGG GCT GTT GGC GAC TCT GCC TAC AGC ACG GGG CAT GTG 1632 Val Trp Tyr Gly Ala Val Gly Asp Ser Ala Tyr Ser Thr Gly His Val 530 535 540 TCC AGG CTG AGC CCG CTG AGC GTC GAC ACA GTC GCT GTT GGC CTG GTG 1680 Ser Arg Leu Ser Pro Leu Ser Val Asp Thr Val Ala Val Gly Leu Val 545 550 555 560 TCC AGC GTG GTT GTC TAT CCC GTC TAC CTG GCC ATC CTT TTT CTC TTC 1728 Ser Ser Val Val Val Tyr Pro Val Tyr Leu Ala Ile Leu Phe Leu Phe 565 570 575 CGG ATG TCC CGG AGC AAG GTG GCT GGG AGC CCG AGC CCC ACA CCT GCC 1776 Arg Met Ser Arg Ser Lys Val Ala Gly Ser Pro Ser Pro Thr Pro Ala 580 585 590 GGG CAG CAG GTG CTG GAC ATC GAC AGC TGC CTG GAC TCG TCC GTG CTG 1824 Gly Gln Gln Val Leu Asp Ile Asp Ser Cys Leu Asp Ser Ser Val Leu 595 600 605 GAC AGC TCC TTC CTC ACG TTC TCA GGC CTC CAC GCT GAG GCC TTT GTT 1872 Asp Ser Ser Phe Leu Thr Phe Ser Gly Leu His Ala Glu Ala Phe Val 610 615 620 GGA CAG ATG AAG AGT GAC TTG TTT CTG GAT GAT TCT AAG AGT CTG GTG 1920 Gly Gln Met Lys Ser Asp Leu Phe Leu Asp Asp Ser Lys Ser Leu Val 625 630 635 640 TGC TGG CCC TCC GGC GAG GGA ACG CTC AGT TGG CCG GAC CTG CTC AGT 1968 Cys Trp Pro Ser Gly Glu Gly Thr Leu Ser Trp Pro Asp Leu Leu Ser 645 650 655 GAC CCG TCC ATT GTG GGT AGC AAT CTG CGG CAG CTG GCA CGG GGC CAG 2016 Asp Pro Ser Ile Val Gly Ser Asn Leu Arg Gln Leu Ala Arg Gly Gln 660 665 670 GCG GGC CAT GGG CTG GGC CCA GAG GAG GAC GGC TTC TCC CTG GCC AGC 2064 Ala Gly His Gly Leu Gly Pro Glu Glu Asp Gly Phe Ser Leu Ala Ser 675 680 685 CCC TAC TCG CCT GCC AAA TCC TTC TCA GCA TCA GAT GAA GAC CTG ATC 2112 Pro Tyr Ser Pro Ala Lys Ser Phe Ser Ala Ser Asp Glu Asp Leu Ile 690 695 700 CAG CAG GTC CTT GCC GAG GGG GTC AGC AGC CCA GCC CCT ACC CAA GAC 2160 Gln Gln Val Leu Ala Glu Gly Val Ser Ser Pro Ala Pro Thr Gln Asp 705 710 715 720 ACC CAC ATG GAA ACG GAC CTG CTC AGC AGC CTG TCC AGC ACT CCT GGG 2208 Thr His Met Glu Thr Asp Leu Leu Ser Ser Leu Ser Ser Thr Pro Gly 725 730 735 GAG AAG ACA GAG ACG CTG GCG CTG CAG AGG CTG GGG GAG CTG GGG CCA 2256 Glu Lys Thr Glu Thr Leu Ala Leu Gln Arg Leu Gly Glu Leu Gly Pro 740 745 750 CCC AGC CCA GGC CTG AAC TGG GAA CAG CCC CAG GCA GCG AGG CTG TCC 2304 Pro Ser Pro Gly Leu Asn Trp Glu Gln Pro Gln Ala Ala Arg Leu Ser 755 760 765 AGG ACA GGA CTG GTG GAG GGT CTG CGG AAG CGC CTG CTG CCG GCC TGG 2352 Arg Thr Gly Leu Val Glu Gly Leu Arg Lys Arg Leu Leu Pro Ala Trp 770 775 780 TGT GCC TCC CTG GCC CAC GGG CTC AGC CTG CTC CTG GTG GCT GTG GCT 2400 Cys Ala Ser Leu Ala His Gly Leu Ser Leu Leu Leu Val Ala Val Ala 785 790 795 800 GTG GCT GTC TCA GGG TGG GTG GGT GCG AGC TTC CCC CCG GGC GTG AGT 2448 Val Ala Val Ser Gly Trp Val Gly Ala Ser Phe Pro Pro Gly Val Ser 805 810 815 GTT GCG TGG CTC CTG TCC AGC AGC GCC AGC TTC CTG GCC TCA TTC CTC 2496 Val Ala Trp Leu Leu Ser Ser Ser Ala Ser Phe Leu Ala Ser Phe Leu 820 825 830 GGC TGG GAG CCA CTG AAG GTC TTG CTG GAA GCC CTG TAC TTC TCA CTG 2544 Gly Trp Glu Pro Leu Lys Val Leu Leu Glu Ala Leu Tyr Phe Ser Leu 835 840 845 GTG GCC AAG CGG CTG CAC CCG GAT GAA GAT GAC ACC CTG GTA GAG AGC 2592 Val Ala Lys Arg Leu His Pro Asp Glu Asp Asp Thr Leu Val Glu Ser 850 855 860 CCG GCT GTG ACG CCT GTG AGC GCA CGT GTG CCC CGC GTA CGG CCA CCC 2640 Pro Ala Val Thr Pro Val Ser Ala Arg Val Pro Arg Val Arg Pro Pro 865 870 875 880 CAC GGC TTT GCA CTC TTC CTG GCC AAG GAA GAA GCC CGC AAG GTC AAG 2688 His Gly Phe Ala Leu Phe Leu Ala Lys Glu Glu Ala Arg Lys Val Lys 885 890 895 AGG CTA CAT GGC ATG CTG CGG AGC CTC CTG GTG TAC ATG CTT TTT CTG 2736 Arg Leu His Gly Met Leu Arg Ser Leu Leu Val Tyr Met Leu Phe Leu 900 905 910 CTG GTG ACC CTG CTG GCC AGC TAT GGG GAT GCC TCA TGC CAT GGG CAC 2784 Leu Val Thr Leu Leu Ala Ser Tyr Gly Asp Ala Ser Cys His Gly His 915 920 925 GCC TAC CGT CTG CAA AGC GCC ATC AAG CAG GAG CTG CAC AGC CGG GCC 2832 Ala Tyr Arg Leu Gln Ser Ala Ile Lys Gln Glu Leu His Ser Arg Ala 930 935 940 TTC CTG GCC ATC ACG CGG TCT GAG GAG CTC TGG CCA TGG ATG GCC CAC 2880 Phe Leu Ala Ile Thr Arg Ser Glu Glu Leu Trp Pro Trp Met Ala His 945 950 955 960 GTG CTG CTG CCC TAC GTC CAC GGG AAC CAG TCC AGC CCA GAG CTG GGG 2928 Val Leu Leu Pro Tyr Val His Gly Asn Gln Ser Ser Pro Glu Leu Gly 965 970 975 CCC CCA CGG CTG CGG CAG GTG CGG CTG CAG GAA GCA CTC TAC CCA GAC 2976 Pro Pro Arg Leu Arg Gln Val Arg Leu Gln Glu Ala Leu Tyr Pro Asp 980 985 990 CCT CCC GGC CCC AGG GTC CAC ACG TGC TCG GCC GCA GGA GGC TTC AGC 3024 Pro Pro Gly Pro Arg Val His Thr Cys Ser Ala Ala Gly Gly Phe Ser 995 1000 1005 ACC AGC GAT TAC GAC GTT GGC TGG GAG AGT CCT CAC AAT GGC TCG GGG 3072 Thr Ser Asp Tyr Asp Val Gly Trp Glu Ser Pro His Asn Gly Ser Gly 1010 1015 1020 ACG TGG GCC TAT TCA GCG CCG GAT CTG CTG GGG GCA TGG TCC TGG GGC 3120 Thr Trp Ala Tyr Ser Ala Pro Asp Leu Leu Gly Ala Trp Ser Trp Gly 1025 1030 1035 1040 TCC TGT GCC GTG TAT GAC AGC GGG GGC TAC GTG CAG GAG CTG GGC CTG 3168 Ser Cys Ala Val Tyr Asp Ser Gly Gly Tyr Val Gln Glu Leu Gly Leu 1045 1050 1055 AGC CTG GAG GAG AGC CGC GAC CGG CTG CGC TTC CTG CAG CTG CAC AAC 3216 Ser Leu Glu Glu Ser Arg Asp Arg Leu Arg Phe Leu Gln Leu His Asn 1060 1065 1070 TGG CTG GAC AAC AGG AGC CGC GCT GTG TTC CTG GAG CTC ACG CGC TAC 3264 Trp Leu Asp Asn Arg Ser Arg Ala Val Phe Leu Glu Leu Thr Arg Tyr 1075 1080 1085 AGC CCG GCC GTG GGG CTG CAC GCC GCC GTC ACG CTG CGC CTC GAG TTC 3312 Ser Pro Ala Val Gly Leu His Ala Ala Val Thr Leu Arg Leu Glu Phe 1090 1095 1100 CCG GCG GCC GGC CGC GCC CTG GCC GCC CTC AGC GTC CGC CCC TTT GCG 3360 Pro Ala Ala Gly Arg Ala Leu Ala Ala Leu Ser Val Arg Pro Phe Ala 1105 1110 1115 1120 CTG CGC CGC CTC AGC GCG GGC CTC TCG CTG CCT CTG CTC ACC TCG GTG 3408 Leu Arg Arg Leu Ser Ala Gly Leu Ser Leu Pro Leu Leu Thr Ser Val 1125 1130 1135 TGC CTG CTG CTG TTC GCC GTG CAC TTC GCC GTG GCC GAG GCC CGT ACT 3456 Cys Leu Leu Leu Phe Ala Val His Phe Ala Val Ala Glu Ala Arg Thr 1140 1145 1150 TGG CAC AGG GAA GGG CGC TGG CGC GTG CTG CGG CTC GGA GCC TGG GCG 3504 Trp His Arg Glu Gly Arg Trp Arg Val Leu Arg Leu Gly Ala Trp Ala 1155 1160 1165 CGG TGG CTG CTG GTG GCG CTG ACG GCG GCC ACG GCA CTG GTA CGC CTC 3552 Arg Trp Leu Leu Val Ala Leu Thr Ala Ala Thr Ala Leu Val Arg Leu 1170 1175 1180 GCC CAG CTG GGT GCC GCT GAC CGC CAG TGG ACC CGT TTC GTG CGC GGC 3600 Ala Gln Leu Gly Ala Ala Asp Arg Gln Trp Thr Arg Phe Val Arg Gly 1185 1190 1195 1200 CGC CCG CGC CGC TTC ACT AGC TTC GAC CAG GTG GCG CAC GTG AGC TCC 3648 Arg Pro Arg Arg Phe Thr Ser Phe Asp Gln Val Ala His Val Ser Ser 1205 1210 1215 GCA GCC CGT GGC CTG GCG GCC TCG CTG CTC TTC CTG CTT TTG GTC AAG 3696 Ala Ala Arg Gly Leu Ala Ala Ser Leu Leu Phe Leu Leu Leu Val Lys 1220 1225 1230 GCT GCC CAG CAC GTA CGC TTC GTG CGC CAG TGG TCC GTC TTT GGC AAG 3744 Ala Ala Gln His Val Arg Phe Val Arg Gln Trp Ser Val Phe Gly Lys 1235 1240 1245 ACA TTA TGC CGA GCT CTG CCA GAG CTC CTG GGG GTC ACC TTG GGC CTG 3792 Thr Leu Cys Arg Ala Leu Pro Glu Leu Leu Gly Val Thr Leu Gly Leu 1250 1255 1260 GTG GTG CTC GGG GTA GCC TAC GCC CAG CTG GCC ATC CTG CTC GTG TCT 3840 Val Val Leu Gly Val Ala Tyr Ala Gln Leu Ala Ile Leu Leu Val Ser 1265 1270 1275 1280 TCC TGT GTG GAC TCC CTC TGG AGC GTG GCC CAG GCC CTG TTG GTG CTG 3888 Ser Cys Val Asp Ser Leu Trp Ser Val Ala Gln Ala Leu Leu Val Leu 1285 1290 1295 TGC CCT GGG ACT GGG CTC TCT ACC CTG TGT CCT GCC GAG TCC TGG CAC 3936 Cys Pro Gly Thr Gly Leu Ser Thr Leu Cys Pro Ala Glu Ser Trp His 1300 1305 1310 CTG TCA CCC CTG CTG TGT GTG GGG CTC TGG GCA CTG CGG CTG TGG GGC 3984 Leu Ser Pro Leu Leu Cys Val Gly Leu Trp Ala Leu Arg Leu Trp Gly 1315 1320 1325 GCC CTA CGG CTG GGG GCT GTT ATT CTC CGC TGG CGC TAC CAC GCC TTG 4032 Ala Leu Arg Leu Gly Ala Val Ile Leu Arg Trp Arg Tyr His Ala Leu 1330 1335 1340 CGT GGA GAG CTG TAC CGG CCG GCC TGG GAG CCC CAG GAC TAC GAG ATG 4080 Arg Gly Glu Leu Tyr Arg Pro Ala Trp Glu Pro Gln Asp Tyr Glu Met 1345 1350 1355 1360 GTG GAG TTG TTC CTG CGC AGG CTG CGC CTC TGG ATG GGC CTC AGC AAG 4128 Val Glu Leu Phe Leu Arg Arg Leu Arg Leu Trp Met Gly Leu Ser Lys 1365 1370 1375 GTC AAG GAG TTC CGC CAC AAA GTC CGC TTT GAA GGG ATG GAG CCG CTG 4176 Val Lys Glu Phe Arg His Lys Val Arg Phe Glu Gly Met Glu Pro Leu 1380 1385 1390 CCC TCT CGC TCC TCC AGG GGC TCC AAG GTA TCC CCG GAT GTG CCC CCA 4224 Pro Ser Arg Ser Ser Arg Gly Ser Lys Val Ser Pro Asp Val Pro Pro 1395 1400 1405 CCC AGC GCT GGC TCC GAT GCC TCG CAC CCC TCC ACC TCC TCC AGC CAG 4272 Pro Ser Ala Gly Ser Asp Ala Ser His Pro Ser Thr Ser Ser Ser Gln 1410 1415 1420 CTG GAT GGG CTG AGC GTG AGC CTG GGC CGG CTG GGG ACA AGG TGT GAG 4320 Leu Asp Gly Leu Ser Val Ser Leu Gly Arg Leu Gly Thr Arg Cys Glu 1425 1430 1435 1440 CCT GAG CCC TCC CGC CTC CAA GCC GTG TTC GAG GCC CTG CTC ACC CAG 4368 Pro Glu Pro Ser Arg Leu Gln Ala Val Phe Glu Ala Leu Leu Thr Gln 1445 1450 1455 TTT GAC CGA CTC AAC CAG GCC ACA GAG GAC GTC TAC CAG CTG GAG CAG 4416 Phe Asp Arg Leu Asn Gln Ala Thr Glu Asp Val Tyr Gln Leu Glu Gln 1460 1465 1470 CAG CTG CAC AGC CTG CAA GGC CGC AGG AGC AGC CGG GCG CCC GCC GGA 4464 Gln Leu His Ser Leu Gln Gly Arg Arg Ser Ser Arg Ala Pro Ala Gly 1475 1480 1485 TCT TCC CGT GGC CCA TCC CCG GGC CTG CGG CCA GCA CTG CCC AGC CGC 4512 Ser Ser Arg Gly Pro Ser Pro Gly Leu Arg Pro Ala Leu Pro Ser Arg 1490 1495 1500 CTT GCC CGG GCC AGT CGG GGT GTG GAC CTG GCC ACT GGC CCC AGC AGG 4560 Leu Ala Arg Ala Ser Arg Gly Val Asp Leu Ala Thr Gly Pro Ser Arg 1505 1510 1515 1520 ACA CCT TCG GGC CAA GAA CAA GGT CCA CCC CAG CAG CAC TTA GTC CTC 4608 Thr Pro Ser Gly Gln Glu Gln Gly Pro Pro Gln Gln His Leu Val Leu 1525 1530 1535 CTT CCT GGC GGG GGT GGG CCG TGG AGT CGG AGT GGA CAC CGC TCA GTA 4656 Leu Pro Gly Gly Gly Gly Pro Trp Ser Arg Ser Gly His Arg Ser Val 1540 1545 1550 TTA CTT TCT GCC GCT GTC AAG GCC GAG GGC CAG GCA GAA TGG CTG CAC 4704 Leu Leu Ser Ala Ala Val Lys Ala Glu Gly Gln Ala Glu Trp Leu His 1555 1560 1565 GTA GGT TCC CCA GAG AGC AGG CAG GGG CAT CTG TCT GTC TGT GGG CTT 4752 Val Gly Ser Pro Glu Ser Arg Gln Gly His Leu Ser Val Cys Gly Leu 1570 1575 1580 CAG CAC TTT AAA GAG GCT GTG TGG CCA ACC AGG ACC CAG GGT CCC CTC 4800 Gln His Phe Lys Glu Ala Val Trp Pro Thr Arg Thr Gln Gly Pro Leu 1585 1590 1595 1600 CCC AGC TCC CTT GGG AAG GAC ACA GCA GTA TTG GAC GGT TTC 4842 Pro Ser Ser Leu Gly Lys Asp Thr Ala Val Leu Asp Gly Phe 1605 1610 TAGCCTCTGA GATGCTAATT TATTTCCCCG AGTCCTCAGG TACAGCGGGC TGTGCCCGGC 4902 CCCACCCCCT GGGCAGATGT CCCCCACTGC TAAGGCTGCT GGCTTCAGGG AGGGTTAGCC 4962 TGCACCGCCG CCACCCTGCC CCTAAGTTAT TACCTCTCCA GTTCCTACCG TACTCCCTGC 5022 ACCGTCTCAC TGTGTGTCTC GTGTCAGTAA TTTATATGGT GTTAAAATGT GTATATTTTT 5082 GTATGTCACT ATTTTCACTA GGGCTGAGGG GCCTGCGCCC AGAGCTGGCC TCCCCCAACA 5142 CCTGCTGCGC TTGGTAGGTG TGGTGGCGTT ATGGCAGCCC GGCTGCTGCT TGGATGCGAG 5202 CTTGGCCTTG GGCCGGTGCT GGGGGCACAG CTGTCTGCCA GGCACTCTCA TCACCCCAGA 5262 GGCCTTGTCA TCCTCCCTTG CCCCAGGCCA GGTAGCAAGA GAGCAGCGCC CAGGCCTGCT 5322 GGCATCAGGT CTGGGCAAGT AGCAGGACTA GGCATGTCAG AGGACCCCAG GGTGGTTAGA 5382 GGAAAAGACT CCTCCTGGGG GCTGGCTCCC AGGGTGGAGG AAGGTGACTG TGTGTGTGTG 5442 TGTGTGCGCG CGCGACGCGC GAGTGTGCTG TATGGCCCAG GCAGCCTCAA GGCCCTCGGA 5502 GCTGGCTGTG CCTGCTTCTG TGTACCACTT CTGTGGGCAT GGCCGCTTCT AGAGCCTCGA 5562 CACCCCCCCA ACCCCCGCAC CAAGCAGACA AAGTCAATAA AAGAGCTGTC TGACTGCAAA 5622 AAAAAAAAA 5631 1614 amino acids amino acid linear protein 2 Leu Asn Glu Glu Pro Leu Thr Leu Ala Gly Glu Glu Ile Val Ala Gln 1 5 10 15 Gly Lys Arg Ser Asp Pro Arg Ser Leu Leu Cys Tyr Gly Gly Ala Pro 20 25 30 Gly Pro Gly Cys His Phe Ser Ile Pro Glu Ala Phe Ser Gly Ala Leu 35 40 45 Ala Asn Leu Ser Asp Val Val Gln Leu Ile Phe Leu Val Asp Ser Asn 50 55 60 Pro Phe Pro Phe Gly Tyr Ile Ser Asn Tyr Thr Val Ser Thr Lys Val 65 70 75 80 Ala Ser Met Ala Phe Gln Thr Gln Ala Gly Ala Gln Ile Pro Ile Glu 85 90 95 Arg Leu Ala Ser Glu Arg Ala Ile Thr Val Lys Val Pro Asn Asn Ser 100 105 110 Asp Trp Ala Ala Arg Gly His Arg Ser Ser Ala Asn Ser Ala Asn Ser 115 120 125 Val Val Val Gln Pro Gln Ala Ser Val Gly Ala Val Val Thr Leu Asp 130 135 140 Ser Ser Asn Pro Ala Ala Gly Leu His Leu Gln Leu Asn Tyr Thr Leu 145 150 155 160 Leu Asp Gly His Tyr Leu Ser Glu Glu Pro Glu Pro Tyr Leu Ala Val 165 170 175 Tyr Leu His Ser Glu Pro Arg Pro Asn Glu His Asn Cys Ser Ala Ser 180 185 190 Arg Arg Ile Arg Pro Glu Ser Leu Gln Gly Ala Asp His Arg Pro Tyr 195 200 205 Thr Phe Phe Ile Ser Pro Gly Ser Arg Asp Pro Ala Gly Ser Tyr His 210 215 220 Leu Asn Leu Ser Ser His Phe Arg Trp Ser Ala Leu Gln Val Ser Val 225 230 235 240 Gly Leu Tyr Thr Ser Leu Cys Gln Tyr Phe Ser Glu Glu Asp Met Val 245 250 255 Trp Arg Thr Glu Gly Leu Leu Pro Leu Glu Glu Thr Ser Pro Arg Gln 260 265 270 Ala Val Cys Leu Thr Arg His Leu Thr Ala Phe Gly Ala Ser Leu Phe 275 280 285 Val Pro Pro Ser His Val Arg Phe Val Phe Pro Glu Pro Thr Ala Asp 290 295 300 Val Asn Tyr Ile Val Met Leu Thr Cys Ala Val Cys Leu Val Thr Tyr 305 310 315 320 Met Val Met Ala Ala Ile Leu His Lys Leu Asp Gln Leu Asp Ala Ser 325 330 335 Arg Gly Arg Ala Ile Pro Phe Cys Gly Gln Arg Gly Arg Phe Lys Tyr 340 345 350 Glu Ile Leu Val Lys Thr Gly Trp Gly Arg Gly Ser Gly Thr Thr Ala 355 360 365 His Val Gly Ile Met Leu Tyr Gly Val Asp Ser Arg Ser Gly His Arg 370 375 380 His Leu Asp Gly Asp Arg Ala Phe His Arg Asn Ser Leu Asp Ile Phe 385 390 395 400 Arg Ile Ala Thr Pro His Ser Leu Gly Ser Val Trp Lys Ile Arg Val 405 410 415 Trp His Asp Asn Lys Gly Leu Ser Pro Ala Trp Phe Leu Gln His Val 420 425 430 Ile Val Arg Asp Leu Gln Thr Ala Arg Ser Ala Phe Phe Leu Val Asn 435 440 445 Asp Trp Leu Ser Val Glu Thr Glu Ala Asn Gly Gly Leu Val Glu Lys 450 455 460 Glu Val Leu Ala Ala Ser Asp Ala Ala Leu Leu Arg Phe Arg Arg Leu 465 470 475 480 Leu Val Ala Glu Leu Gln Arg Gly Phe Phe Asp Lys His Ile Trp Leu 485 490 495 Ser Ile Trp Asp Arg Pro Pro Arg Ser Arg Phe Thr Arg Ile Gln Arg 500 505 510 Ala Thr Cys Cys Val Leu Leu Ile Cys Leu Phe Leu Gly Ala Asn Ala 515 520 525 Val Trp Tyr Gly Ala Val Gly Asp Ser Ala Tyr Ser Thr Gly His Val 530 535 540 Ser Arg Leu Ser Pro Leu Ser Val Asp Thr Val Ala Val Gly Leu Val 545 550 555 560 Ser Ser Val Val Val Tyr Pro Val Tyr Leu Ala Ile Leu Phe Leu Phe 565 570 575 Arg Met Ser Arg Ser Lys Val Ala Gly Ser Pro Ser Pro Thr Pro Ala 580 585 590 Gly Gln Gln Val Leu Asp Ile Asp Ser Cys Leu Asp Ser Ser Val Leu 595 600 605 Asp Ser Ser Phe Leu Thr Phe Ser Gly Leu His Ala Glu Ala Phe Val 610 615 620 Gly Gln Met Lys Ser Asp Leu Phe Leu Asp Asp Ser Lys Ser Leu Val 625 630 635 640 Cys Trp Pro Ser Gly Glu Gly Thr Leu Ser Trp Pro Asp Leu Leu Ser 645 650 655 Asp Pro Ser Ile Val Gly Ser Asn Leu Arg Gln Leu Ala Arg Gly Gln 660 665 670 Ala Gly His Gly Leu Gly Pro Glu Glu Asp Gly Phe Ser Leu Ala Ser 675 680 685 Pro Tyr Ser Pro Ala Lys Ser Phe Ser Ala Ser Asp Glu Asp Leu Ile 690 695 700 Gln Gln Val Leu Ala Glu Gly Val Ser Ser Pro Ala Pro Thr Gln Asp 705 710 715 720 Thr His Met Glu Thr Asp Leu Leu Ser Ser Leu Ser Ser Thr Pro Gly 725 730 735 Glu Lys Thr Glu Thr Leu Ala Leu Gln Arg Leu Gly Glu Leu Gly Pro 740 745 750 Pro Ser Pro Gly Leu Asn Trp Glu Gln Pro Gln Ala Ala Arg Leu Ser 755 760 765 Arg Thr Gly Leu Val Glu Gly Leu Arg Lys Arg Leu Leu Pro Ala Trp 770 775 780 Cys Ala Ser Leu Ala His Gly Leu Ser Leu Leu Leu Val Ala Val Ala 785 790 795 800 Val Ala Val Ser Gly Trp Val Gly Ala Ser Phe Pro Pro Gly Val Ser 805 810 815 Val Ala Trp Leu Leu Ser Ser Ser Ala Ser Phe Leu Ala Ser Phe Leu 820 825 830 Gly Trp Glu Pro Leu Lys Val Leu Leu Glu Ala Leu Tyr Phe Ser Leu 835 840 845 Val Ala Lys Arg Leu His Pro Asp Glu Asp Asp Thr Leu Val Glu Ser 850 855 860 Pro Ala Val Thr Pro Val Ser Ala Arg Val Pro Arg Val Arg Pro Pro 865 870 875 880 His Gly Phe Ala Leu Phe Leu Ala Lys Glu Glu Ala Arg Lys Val Lys 885 890 895 Arg Leu His Gly Met Leu Arg Ser Leu Leu Val Tyr Met Leu Phe Leu 900 905 910 Leu Val Thr Leu Leu Ala Ser Tyr Gly Asp Ala Ser Cys His Gly His 915 920 925 Ala Tyr Arg Leu Gln Ser Ala Ile Lys Gln Glu Leu His Ser Arg Ala 930 935 940 Phe Leu Ala Ile Thr Arg Ser Glu Glu Leu Trp Pro Trp Met Ala His 945 950 955 960 Val Leu Leu Pro Tyr Val His Gly Asn Gln Ser Ser Pro Glu Leu Gly 965 970 975 Pro Pro Arg Leu Arg Gln Val Arg Leu Gln Glu Ala Leu Tyr Pro Asp 980 985 990 Pro Pro Gly Pro Arg Val His Thr Cys Ser Ala Ala Gly Gly Phe Ser 995 1000 1005 Thr Ser Asp Tyr Asp Val Gly Trp Glu Ser Pro His Asn Gly Ser Gly 1010 1015 1020 Thr Trp Ala Tyr Ser Ala Pro Asp Leu Leu Gly Ala Trp Ser Trp Gly 1025 1030 1035 1040 Ser Cys Ala Val Tyr Asp Ser Gly Gly Tyr Val Gln Glu Leu Gly Leu 1045 1050 1055 Ser Leu Glu Glu Ser Arg Asp Arg Leu Arg Phe Leu Gln Leu His Asn 1060 1065 1070 Trp Leu Asp Asn Arg Ser Arg Ala Val Phe Leu Glu Leu Thr Arg Tyr 1075 1080 1085 Ser Pro Ala Val Gly Leu His Ala Ala Val Thr Leu Arg Leu Glu Phe 1090 1095 1100 Pro Ala Ala Gly Arg Ala Leu Ala Ala Leu Ser Val Arg Pro Phe Ala 1105 1110 1115 1120 Leu Arg Arg Leu Ser Ala Gly Leu Ser Leu Pro Leu Leu Thr Ser Val 1125 1130 1135 Cys Leu Leu Leu Phe Ala Val His Phe Ala Val Ala Glu Ala Arg Thr 1140 1145 1150 Trp His Arg Glu Gly Arg Trp Arg Val Leu Arg Leu Gly Ala Trp Ala 1155 1160 1165 Arg Trp Leu Leu Val Ala Leu Thr Ala Ala Thr Ala Leu Val Arg Leu 1170 1175 1180 Ala Gln Leu Gly Ala Ala Asp Arg Gln Trp Thr Arg Phe Val Arg Gly 1185 1190 1195 1200 Arg Pro Arg Arg Phe Thr Ser Phe Asp Gln Val Ala His Val Ser Ser 1205 1210 1215 Ala Ala Arg Gly Leu Ala Ala Ser Leu Leu Phe Leu Leu Leu Val Lys 1220 1225 1230 Ala Ala Gln His Val Arg Phe Val Arg Gln Trp Ser Val Phe Gly Lys 1235 1240 1245 Thr Leu Cys Arg Ala Leu Pro Glu Leu Leu Gly Val Thr Leu Gly Leu 1250 1255 1260 Val Val Leu Gly Val Ala Tyr Ala Gln Leu Ala Ile Leu Leu Val Ser 1265 1270 1275 1280 Ser Cys Val Asp Ser Leu Trp Ser Val Ala Gln Ala Leu Leu Val Leu 1285 1290 1295 Cys Pro Gly Thr Gly Leu Ser Thr Leu Cys Pro Ala Glu Ser Trp His 1300 1305 1310 Leu Ser Pro Leu Leu Cys Val Gly Leu Trp Ala Leu Arg Leu Trp Gly 1315 1320 1325 Ala Leu Arg Leu Gly Ala Val Ile Leu Arg Trp Arg Tyr His Ala Leu 1330 1335 1340 Arg Gly Glu Leu Tyr Arg Pro Ala Trp Glu Pro Gln Asp Tyr Glu Met 1345 1350 1355 1360 Val Glu Leu Phe Leu Arg Arg Leu Arg Leu Trp Met Gly Leu Ser Lys 1365 1370 1375 Val Lys Glu Phe Arg His Lys Val Arg Phe Glu Gly Met Glu Pro Leu 1380 1385 1390 Pro Ser Arg Ser Ser Arg Gly Ser Lys Val Ser Pro Asp Val Pro Pro 1395 1400 1405 Pro Ser Ala Gly Ser Asp Ala Ser His Pro Ser Thr Ser Ser Ser Gln 1410 1415 1420 Leu Asp Gly Leu Ser Val Ser Leu Gly Arg Leu Gly Thr Arg Cys Glu 1425 1430 1435 1440 Pro Glu Pro Ser Arg Leu Gln Ala Val Phe Glu Ala Leu Leu Thr Gln 1445 1450 1455 Phe Asp Arg Leu Asn Gln Ala Thr Glu Asp Val Tyr Gln Leu Glu Gln 1460 1465 1470 Gln Leu His Ser Leu Gln Gly Arg Arg Ser Ser Arg Ala Pro Ala Gly 1475 1480 1485 Ser Ser Arg Gly Pro Ser Pro Gly Leu Arg Pro Ala Leu Pro Ser Arg 1490 1495 1500 Leu Ala Arg Ala Ser Arg Gly Val Asp Leu Ala Thr Gly Pro Ser Arg 1505 1510 1515 1520 Thr Pro Ser Gly Gln Glu Gln Gly Pro Pro Gln Gln His Leu Val Leu 1525 1530 1535 Leu Pro Gly Gly Gly Gly Pro Trp Ser Arg Ser Gly His Arg Ser Val 1540 1545 1550 Leu Leu Ser Ala Ala Val Lys Ala Glu Gly Gln Ala Glu Trp Leu His 1555 1560 1565 Val Gly Ser Pro Glu Ser Arg Gln Gly His Leu Ser Val Cys Gly Leu 1570 1575 1580 Gln His Phe Lys Glu Ala Val Trp Pro Thr Arg Thr Gln Gly Pro Leu 1585 1590 1595 1600 Pro Ser Ser Leu Gly Lys Asp Thr Ala Val Leu Asp Gly Phe 1605 1610 553 base pairs nucleic acid unknown unknown cDNA Homo sapiens misc_feature 1..533 /function= “1A1 H.6 probe” 3 AGCTTGGCAC CATCAAGGGC CAGTTCAACT TTGTCCACGT GATCGTCACC CCGCTGGACT 60 ACGAGTGCAA CCTGGTGTCC CTGCAGTGCA GGAAAGACAT GGAGGGCCTT GTGGACACCA 120 GCGTGGCCAA GATCGTGTCT GACCGCAACC TGCCCTTCGT GGCCCGCCAG ATGGCCCTGC 180 ACGCAAATAT GGCCTCACAG GTGCATCATA GCCGCTCCAA CCCCACCGAT ATCTACCCCT 240 CCAAGTGGAT TGCCCGGCTC CGCCACATCA AGCGGCTCCG CCAGCGGATC TGCGAGGAAG 300 CCGCCTACTC CAACCCCAGC CTACCTCTGG TGCACCCTCC GTCCCATAGC AAAGCCCCTG 360 CACAGACTCC AGCCGAGCCC ACACCTGGCT ATGAGGTGGG CCAGCGGAAG CGCCTCATCT 420 CCTCGGTGGA GGACTTCACC GAGTTTGTGT GAGGCCGGGG CCCTCCCTCC TGCACTGGCC 480 TTGGACGGTA TTGCCTGTCA GTGAAATAAA TAAAGTCCTG ACCCCAGTGC ACAGACATAG 540 AGGCACAGAT TGC 553 517 base pairs nucleic acid unknown unknown DNA (genomic) Homo sapiens misc_feature 1..517 /function= “CW10 probe” 4 CTGGTGTGTG TGAGACGTGC GGGGCTGGGA AGTGTTGGCA GAGCCGCGAG TACCGTCCTC 60 ACTCCTTTTG TTCTTTTGAC GTAAGCTGGC GAGTGGCACT GCCTGAGTTC CGCTCAGTGC 120 CCGCCCTGAT GTGCGGACCC CGCTGCATTC TTGCTGTTAG GTGGTGGCGG TGTGCGCTGT 180 CGCTGGTGGG CACCGAGAGT CTTTGGGAGC TTTGGGGAGG TTGTGCCAAG CCTGAGCCTC 240 GACGTCCCCC TTCCCGGCTT TCTGTTGGCT CTTCTGAGGC CAGGGCATCT CTATGAGGGC 300 CTCCTGCTGG AGCCGTCTCT GTGGATCTCC TCTGCCATCC TGGCCCATGA GTGGGTGATG 360 CGCTGGCCAC CATCTGGTGA CAGTGGCCGG GCACCGCTGC CAAATGTGGG TCCCGCATCT 420 GCAAGCCCCT CCCTGGGTCC CCTAGGGTAT GGGGTGGTTC TGCCACTGCC CTCGCTCCCC 480 CACCTTGGGG TGCCTCTCCC CCTGCTCGTG GGGGAGA 517 13807 base pairs nucleic acid unknown unknown cDNA Homo sapiens CDS 2..13018 misc_feature 7295..8184 /function= “g alpha 22 fragment” misc_feature 6422..7294 /function= “GAP GAMMA PETER fragment” misc_feature 3697..6421 /function= “JH8 fragment” misc_feature 1373..1701 /function= “S3/S4 PETER fragment” misc_feature 2176..2962 /function= “S3/S4 CON2 PETER fragment” misc_feature 2963..3696 /function= “S1/S3 PETER fragment” misc_feature 118..1372 /function= “S4/JH13 fragment” misc_feature 1..85 /function= “5′ COMPLETE [Split] fragment” misc_feature 87..3696 /function= “5′ COMPLETE [Split] fragment” misc_feature 1..85 /function= “6 (5) R cDNA [Split] fragment” misc_feature 87..117 /product= “6 (5) R cDNA [Split] fragment” 5 C GGC GCC GCC TGC CGC GTC AAC TGC TCG GGC CGC GGG CTG CGG ACG 46 Gly Ala Ala Cys Arg Val Asn Cys Ser Gly Arg Gly Leu Arg Thr 1615 1620 1625 CTC GGT CCC GCG CTG CGC ATC CCC GCG GAC GCC ACA GCG CTA GAC GTC 94 Leu Gly Pro Ala Leu Arg Ile Pro Ala Asp Ala Thr Ala Leu Asp Val 1630 1635 1640 1645 TCC CAC AAC CTG CTC CGG GCG CTG GAC GTT GGG CTC CTG GCG AAC CTC 142 Ser His Asn Leu Leu Arg Ala Leu Asp Val Gly Leu Leu Ala Asn Leu 1650 1655 1660 TCG GCG CTG GCA GAG CTG GAT ATA AGC AAC AAC AAG ATT TCT ACG TTA 190 Ser Ala Leu Ala Glu Leu Asp Ile Ser Asn Asn Lys Ile Ser Thr Leu 1665 1670 1675 GAA GAA GGA ATA TTT GCT AAT TTA TTT AAT TTA AGT GAA ATA AAC CTG 238 Glu Glu Gly Ile Phe Ala Asn Leu Phe Asn Leu Ser Glu Ile Asn Leu 1680 1685 1690 AGT GGG AAC CCG TTT GAG TGT GAC TGT GGC CTG GCG TGG CTG CCG CGA 286 Ser Gly Asn Pro Phe Glu Cys Asp Cys Gly Leu Ala Trp Leu Pro Arg 1695 1700 1705 TGG GCG GAG GAG CAG CAG GTG CGG GTG GTG CAG CCC GAG GCA GCC ACG 334 Trp Ala Glu Glu Gln Gln Val Arg Val Val Gln Pro Glu Ala Ala Thr 1710 1715 1720 1725 TGT GCT GGG CCT GGC TCC CTG GCT GGC CAG CCT CTG CTT GGC ATC CCC 382 Cys Ala Gly Pro Gly Ser Leu Ala Gly Gln Pro Leu Leu Gly Ile Pro 1730 1735 1740 TTG CTG GAC AGT GGC TGT GGT GAG GAG TAT GTC GCC TGC CTC CCT GAC 430 Leu Leu Asp Ser Gly Cys Gly Glu Glu Tyr Val Ala Cys Leu Pro Asp 1745 1750 1755 AAC AGC TCA GGC ACC GTG GCA GCA GTG TCC TTT TCA GCT GCC CAC GAA 478 Asn Ser Ser Gly Thr Val Ala Ala Val Ser Phe Ser Ala Ala His Glu 1760 1765 1770 GGC CTG CTT CAG CCA GAG GCC TGC AGC GCC TTC TGC TTC TCC ACC GGC 526 Gly Leu Leu Gln Pro Glu Ala Cys Ser Ala Phe Cys Phe Ser Thr Gly 1775 1780 1785 CAG GGC CTC GCA GCC CTC TCG GAG CAG GGC TGG TGC CTG TGT GGG GCG 574 Gln Gly Leu Ala Ala Leu Ser Glu Gln Gly Trp Cys Leu Cys Gly Ala 1790 1795 1800 1805 GCC CAG CCC TCC AGT GCC TCC TTT GCC TGC CTG TCC CTC TGC TCC GGC 622 Ala Gln Pro Ser Ser Ala Ser Phe Ala Cys Leu Ser Leu Cys Ser Gly 1810 1815 1820 CCC CCG CCA CCT CCT GCC CCC ACC TGT AGG GGC CCC ACC CTC CTC CAG 670 Pro Pro Pro Pro Pro Ala Pro Thr Cys Arg Gly Pro Thr Leu Leu Gln 1825 1830 1835 CAC GTC TTC CCT GCC TCC CCA GGG GCC ACC CTG GTG GGG CCC CAC GGA 718 His Val Phe Pro Ala Ser Pro Gly Ala Thr Leu Val Gly Pro His Gly 1840 1845 1850 CCT CTG GCC TCT GGC CAG CTA GCA GCC TTC CAC ATC GCT GCC CCG CTC 766 Pro Leu Ala Ser Gly Gln Leu Ala Ala Phe His Ile Ala Ala Pro Leu 1855 1860 1865 CCT GTC ACT GCC ACA CGC TGG GAC TTC GGA GAC GGC TCC GCC GAG GTG 814 Pro Val Thr Ala Thr Arg Trp Asp Phe Gly Asp Gly Ser Ala Glu Val 1870 1875 1880 1885 GAT GCC GCT GGG CCG GCT GCC TCG CAT CGC TAT GTG CTG CCT GGG CGC 862 Asp Ala Ala Gly Pro Ala Ala Ser His Arg Tyr Val Leu Pro Gly Arg 1890 1895 1900 TAT CAC GTG ACG GCC GTG CTG GCC CTG GGG GCC GGC TCA GCC CTG CTG 910 Tyr His Val Thr Ala Val Leu Ala Leu Gly Ala Gly Ser Ala Leu Leu 1905 1910 1915 GGG ACA GAC GTG CAG GTG GAA GCG GCA CCT GCC GCC CTG GAG CTC GTG 958 Gly Thr Asp Val Gln Val Glu Ala Ala Pro Ala Ala Leu Glu Leu Val 1920 1925 1930 TGC CCG TCC TCG GTG CAG AGT GAC GAG AGC CTT GAC CTC AGC ATC CAG 1006 Cys Pro Ser Ser Val Gln Ser Asp Glu Ser Leu Asp Leu Ser Ile Gln 1935 1940 1945 AAC CGC GGT GGT TCA GGC CTG GAG GCC GCC TAC AGC ATC GTG GCC CTG 1054 Asn Arg Gly Gly Ser Gly Leu Glu Ala Ala Tyr Ser Ile Val Ala Leu 1950 1955 1960 1965 GGC GAG GAG CCG GCC CGA GCG GTG CAC CCG CTC TGC CCC TCG GAC ACG 1102 Gly Glu Glu Pro Ala Arg Ala Val His Pro Leu Cys Pro Ser Asp Thr 1970 1975 1980 GAG ATC TTC CCT GGC AAC GGG CAC TGC TAC CGC CTG GTG GTG GAG AAG 1150 Glu Ile Phe Pro Gly Asn Gly His Cys Tyr Arg Leu Val Val Glu Lys 1985 1990 1995 GCG GCC TGG CTG CAG GCG CAG GAG CAG TGT CAG GCC TGG GCC GGG GCC 1198 Ala Ala Trp Leu Gln Ala Gln Glu Gln Cys Gln Ala Trp Ala Gly Ala 2000 2005 2010 GCC CTG GCA ATG GTG GAC AGT CCC GCC GTG CAG CGC TTC CTG GTC TCC 1246 Ala Leu Ala Met Val Asp Ser Pro Ala Val Gln Arg Phe Leu Val Ser 2015 2020 2025 CGG GTC ACC AGG AGC CTA GAC GTG TGG ATC GGC TTC TCG ACT GTG CAG 1294 Arg Val Thr Arg Ser Leu Asp Val Trp Ile Gly Phe Ser Thr Val Gln 2030 2035 2040 2045 GGG GTG GAG GTG GGC CCA GCG CCG CAG GGC GAG GCC TTC AGC CTG GAG 1342 Gly Val Glu Val Gly Pro Ala Pro Gln Gly Glu Ala Phe Ser Leu Glu 2050 2055 2060 AGC TGC CAG AAC TGG CTG CCC GGG GAG CCA CAC CCA GCC ACA GCC GAG 1390 Ser Cys Gln Asn Trp Leu Pro Gly Glu Pro His Pro Ala Thr Ala Glu 2065 2070 2075 CAC TGC GTC CGG CTC GGG CCC ACC GGG TGG TGT AAC ACC GAC CTG TGC 1438 His Cys Val Arg Leu Gly Pro Thr Gly Trp Cys Asn Thr Asp Leu Cys 2080 2085 2090 TCA GCG CCG CAC AGC TAC GTC TGC GAG CTG CAG CCC GGA GGC CCA GTG 1486 Ser Ala Pro His Ser Tyr Val Cys Glu Leu Gln Pro Gly Gly Pro Val 2095 2100 2105 CAG GAT GCC GAG AAC CTC CTC GTG GGA GCG CCC AGT GGG GAC CTG CAG 1534 Gln Asp Ala Glu Asn Leu Leu Val Gly Ala Pro Ser Gly Asp Leu Gln 2110 2115 2120 2125 GGA CCC CTG ACG CCT CTG GCA CAG CAG GAC GGC CTC TCA GCC CCG CAC 1582 Gly Pro Leu Thr Pro Leu Ala Gln Gln Asp Gly Leu Ser Ala Pro His 2130 2135 2140 GAG CCC GTG GAG GTC ATG GTA TTC CCG GGC CTG CGT CTG AGC CGT GAA 1630 Glu Pro Val Glu Val Met Val Phe Pro Gly Leu Arg Leu Ser Arg Glu 2145 2150 2155 GCC TTC CTC ACC ACG GCC GAA TTT GGG ACC CAG GAG CTC CGG CGG CCC 1678 Ala Phe Leu Thr Thr Ala Glu Phe Gly Thr Gln Glu Leu Arg Arg Pro 2160 2165 2170 GCC CAG CTG CGG CTG CAG GTG TAC CGG CTC CTC AGC ACA GCA GGG ACC 1726 Ala Gln Leu Arg Leu Gln Val Tyr Arg Leu Leu Ser Thr Ala Gly Thr 2175 2180 2185 CCG GAG AAC GGC AGC GAG CCT GAG AGC AGG TCC CCG GAC AAC AGG ACC 1774 Pro Glu Asn Gly Ser Glu Pro Glu Ser Arg Ser Pro Asp Asn Arg Thr 2190 2195 2200 2205 CAG CTG GCC CCC GCG TGC ATG CCA GGG GGA CGC TGG TGC CCT GGA GCC 1822 Gln Leu Ala Pro Ala Cys Met Pro Gly Gly Arg Trp Cys Pro Gly Ala 2210 2215 2220 AAC ATC TGC TTG CCG CTG GAC GCC TCT TGC CAC CCC CAG GCC TGC GCC 1870 Asn Ile Cys Leu Pro Leu Asp Ala Ser Cys His Pro Gln Ala Cys Ala 2225 2230 2235 AAT GGC TGC ACG TCA GGG CCA GGG CTA CCC GGG GCC CCC TAT GCG CTA 1918 Asn Gly Cys Thr Ser Gly Pro Gly Leu Pro Gly Ala Pro Tyr Ala Leu 2240 2245 2250 TGG AGA GAG TTC CTC TTC TCC GTT GCC GCG GGG CCC CCC GCG CAG TAC 1966 Trp Arg Glu Phe Leu Phe Ser Val Ala Ala Gly Pro Pro Ala Gln Tyr 2255 2260 2265 TCG GTC ACC CTC CAC GGC CAG GAT GTC CTC ATG CTC CCT GGT GAC CTC 2014 Ser Val Thr Leu His Gly Gln Asp Val Leu Met Leu Pro Gly Asp Leu 2270 2275 2280 2285 GTT GGC TTG CAG CAC GAC GCT GGC CCT GGC GCC CTC CTG CAC TGC TCG 2062 Val Gly Leu Gln His Asp Ala Gly Pro Gly Ala Leu Leu His Cys Ser 2290 2295 2300 CCG GCT CCC GGC CAC CCT GGT CCC CAG GCC CCG TAC CTC TCC GCC AAC 2110 Pro Ala Pro Gly His Pro Gly Pro Gln Ala Pro Tyr Leu Ser Ala Asn 2305 2310 2315 GCC TCG TCA TGG CTG CCC CAC TTG CCA GCC CAG CTG GAG GGC ACT TGG 2158 Ala Ser Ser Trp Leu Pro His Leu Pro Ala Gln Leu Glu Gly Thr Trp 2320 2325 2330 GCC TGC CCT GCC TGT GCC CTG CGG CTG CTT GCA GCC ACG GAA CAG CTC 2206 Ala Cys Pro Ala Cys Ala Leu Arg Leu Leu Ala Ala Thr Glu Gln Leu 2335 2340 2345 ACC GTG CTG CTG GGC TTG AGG CCC AAC CCT GGA CTG CGG ATG CCT GGG 2254 Thr Val Leu Leu Gly Leu Arg Pro Asn Pro Gly Leu Arg Met Pro Gly 2350 2355 2360 2365 CGC TAT GAG GTC CGG GCA GAG GTG GGC AAT GGC GTG TCC AGG CAC AAC 2302 Arg Tyr Glu Val Arg Ala Glu Val Gly Asn Gly Val Ser Arg His Asn 2370 2375 2380 CTC TCC TGC AGC TTT GAC GTG GTC TCC CCA GTG GCT GGG CTG CGG GTC 2350 Leu Ser Cys Ser Phe Asp Val Val Ser Pro Val Ala Gly Leu Arg Val 2385 2390 2395 ATC TAC CCT GCC CCC CGC GAC GGC CGC CTC TAC GTG CCC ACC AAC GGC 2398 Ile Tyr Pro Ala Pro Arg Asp Gly Arg Leu Tyr Val Pro Thr Asn Gly 2400 2405 2410 TCA GCC TTG GTG CTC CAG GTG GAC TCT GGT GCC AAC GCC ACG GCC ACG 2446 Ser Ala Leu Val Leu Gln Val Asp Ser Gly Ala Asn Ala Thr Ala Thr 2415 2420 2425 GCT CGC TGG CCT GGG GGC AGT GTC AGC GCC CGC TTT GAG AAT GTC TGC 2494 Ala Arg Trp Pro Gly Gly Ser Val Ser Ala Arg Phe Glu Asn Val Cys 2430 2435 2440 2445 CCT GCC CTG GTG GCC ACC TTC GTG CCC GGC TGC CCC TGG GAG ACC AAC 2542 Pro Ala Leu Val Ala Thr Phe Val Pro Gly Cys Pro Trp Glu Thr Asn 2450 2455 2460 GAT ACC CTG TTC TCA GTG GTA GCA CTG CCG TGG CTC AGT GAG GGG GAG 2590 Asp Thr Leu Phe Ser Val Val Ala Leu Pro Trp Leu Ser Glu Gly Glu 2465 2470 2475 CAC GTG GTG GAC GTG GTG GTG GAA AAC AGC GCC AGC CGG GCC AAC CTC 2638 His Val Val Asp Val Val Val Glu Asn Ser Ala Ser Arg Ala Asn Leu 2480 2485 2490 AGC CTG CGG GTG ACG GCG GAG GAG CCC ATC TGT GGC CTC CGC GCC ACG 2686 Ser Leu Arg Val Thr Ala Glu Glu Pro Ile Cys Gly Leu Arg Ala Thr 2495 2500 2505 CCC AGC CCC GAG GCC CGT GTA CTG CAG GGA GTC CTA GTG AGG TAC AGC 2734 Pro Ser Pro Glu Ala Arg Val Leu Gln Gly Val Leu Val Arg Tyr Ser 2510 2515 2520 2525 CCC GTG GTG GAG GCC GGC TCG GAC ATG GTC TTC CGG TGG ACC ATC AAC 2782 Pro Val Val Glu Ala Gly Ser Asp Met Val Phe Arg Trp Thr Ile Asn 2530 2535 2540 GAC AAG CAG TCC CTG ACC TTC CAG AAC GTG GTC TTC AAT GTC ATT TAT 2830 Asp Lys Gln Ser Leu Thr Phe Gln Asn Val Val Phe Asn Val Ile Tyr 2545 2550 2555 CAG AGC GCG GCG GTC TTC AAG CTC TCA CTG ACG GCC TCC AAC CAC GTG 2878 Gln Ser Ala Ala Val Phe Lys Leu Ser Leu Thr Ala Ser Asn His Val 2560 2565 2570 AGC AAC GTC ACC GTG AAC TAC AAC GTA ACC GTG GAG CGG ATG AAC AGG 2926 Ser Asn Val Thr Val Asn Tyr Asn Val Thr Val Glu Arg Met Asn Arg 2575 2580 2585 ATG CAG GGT CTG CAG GTC TCC ACA GTG CCG GCC GTG CTG TCC CCC AAT 2974 Met Gln Gly Leu Gln Val Ser Thr Val Pro Ala Val Leu Ser Pro Asn 2590 2595 2600 2605 GCC ACA CTG GTA CTG ACG GGT GGT GTG CTG GTG GAC TCA GCT GTG GAG 3022 Ala Thr Leu Val Leu Thr Gly Gly Val Leu Val Asp Ser Ala Val Glu 2610 2615 2620 GTG GCC TTC CTG TGG AAC TTT GGG GAT GGG GAG CAG GCC CTC CAC CAG 3070 Val Ala Phe Leu Trp Asn Phe Gly Asp Gly Glu Gln Ala Leu His Gln 2625 2630 2635 TTC CAG CCT CCG TAC AAC GAG TCC TTC CCG GTT CCA GAC CCC TCG GTG 3118 Phe Gln Pro Pro Tyr Asn Glu Ser Phe Pro Val Pro Asp Pro Ser Val 2640 2645 2650 GCC CAG GTG CTG GTG GAG CAC AAT GTC ATG CAC ACC TAC GCT GCC CCA 3166 Ala Gln Val Leu Val Glu His Asn Val Met His Thr Tyr Ala Ala Pro 2655 2660 2665 GGT GAG TAC CTC CTG ACC GTG CTG GCA TCT AAT GCC TTC GAG AAC CTG 3214 Gly Glu Tyr Leu Leu Thr Val Leu Ala Ser Asn Ala Phe Glu Asn Leu 2670 2675 2680 2685 ACG CAG CAG GTG CCT GTG AGC GTG CGC GCC TCC CTG CCC TCC GTG GCT 3262 Thr Gln Gln Val Pro Val Ser Val Arg Ala Ser Leu Pro Ser Val Ala 2690 2695 2700 GTG GGT GTG AGT GAC GGC GTC CTG GTG GCC GGC CGG CCC GTC ACC TTC 3310 Val Gly Val Ser Asp Gly Val Leu Val Ala Gly Arg Pro Val Thr Phe 2705 2710 2715 TAC CCG CAC CCG CTG CCC TCG CCT GGG GGT GTT CTT TAC ACG TGG GAC 3358 Tyr Pro His Pro Leu Pro Ser Pro Gly Gly Val Leu Tyr Thr Trp Asp 2720 2725 2730 TTC GGG GAC GGC TCC CCT GTC CTG ACC CAG AGC CAG CCG GCT GCC AAC 3406 Phe Gly Asp Gly Ser Pro Val Leu Thr Gln Ser Gln Pro Ala Ala Asn 2735 2740 2745 CAC ACC TAT GCC TCG AGG GGC ACC TAC CAC GTG CGC CTG GAG GTC AAC 3454 His Thr Tyr Ala Ser Arg Gly Thr Tyr His Val Arg Leu Glu Val Asn 2750 2755 2760 2765 AAC ACG GTG AGC GGT GCG GCG GCC CAG GCG GAT GTG CGC GTC TTT GAG 3502 Asn Thr Val Ser Gly Ala Ala Ala Gln Ala Asp Val Arg Val Phe Glu 2770 2775 2780 GAG CTC CGC GGA CTC AGC GTG GAC ATG AGC CTG GCC GTG GAG CAG GGC 3550 Glu Leu Arg Gly Leu Ser Val Asp Met Ser Leu Ala Val Glu Gln Gly 2785 2790 2795 GCC CCC GTG GTG GTC AGC GCC GCG GTG CAG ACG GGC GAC AAC ATC ACG 3598 Ala Pro Val Val Val Ser Ala Ala Val Gln Thr Gly Asp Asn Ile Thr 2800 2805 2810 TGG ACC TTC GAC ATG GGG GAC GGC ACC GTG CTG TCG GGC CCG GAG GCA 3646 Trp Thr Phe Asp Met Gly Asp Gly Thr Val Leu Ser Gly Pro Glu Ala 2815 2820 2825 ACA GTG GAG CAT GTG TAC CTG CGG GCA CAG AAC TGC ACA GTG ACC GTG 3694 Thr Val Glu His Val Tyr Leu Arg Ala Gln Asn Cys Thr Val Thr Val 2830 2835 2840 2845 GGT GCG GCC AGC CCC GCC GGC CAC CTG GCC CGG AGC CTG CAC GTG CTG 3742 Gly Ala Ala Ser Pro Ala Gly His Leu Ala Arg Ser Leu His Val Leu 2850 2855 2860 GTC TTC GTC CTG GAG GTG CTG CGC GTT GAA CCC GCC GCC TGC ATC CCC 3790 Val Phe Val Leu Glu Val Leu Arg Val Glu Pro Ala Ala Cys Ile Pro 2865 2870 2875 ACG CAG CCT GAC GCG CGG CTC ACG GCC TAC GTC ACC GGG AAC CCG GCC 3838 Thr Gln Pro Asp Ala Arg Leu Thr Ala Tyr Val Thr Gly Asn Pro Ala 2880 2885 2890 CAC TAC CTC TTC GAC TGG ACC TTC GGG GAT GGC TCC TCC AAC ACG ACC 3886 His Tyr Leu Phe Asp Trp Thr Phe Gly Asp Gly Ser Ser Asn Thr Thr 2895 2900 2905 GTG CGG GGG TGC CCG ACG GTG ACA CAC AAC TTC ACG CGG AGC GGC ACG 3934 Val Arg Gly Cys Pro Thr Val Thr His Asn Phe Thr Arg Ser Gly Thr 2910 2915 2920 2925 TTC CCC CTG GCG CTG GTG CTG TCC AGC CGC GTG AAC AGG GCG CAT TAC 3982 Phe Pro Leu Ala Leu Val Leu Ser Ser Arg Val Asn Arg Ala His Tyr 2930 2935 2940 TTC ACC AGC ATC TGC GTG GAG CCA GAG GTG GGC AAC GTC ACC CTG CAG 4030 Phe Thr Ser Ile Cys Val Glu Pro Glu Val Gly Asn Val Thr Leu Gln 2945 2950 2955 CCA GAG AGG CAG TTT GTG CAG CTC GGG GAC GAG GCC TGG CTG GTG GCA 4078 Pro Glu Arg Gln Phe Val Gln Leu Gly Asp Glu Ala Trp Leu Val Ala 2960 2965 2970 TGT GCC TGG CCC CCG TTC CCC TAC CGC TAC ACC TGG GAC TTT GGC ACC 4126 Cys Ala Trp Pro Pro Phe Pro Tyr Arg Tyr Thr Trp Asp Phe Gly Thr 2975 2980 2985 GAG GAA GCC GCC CCC ACC CGT GCC AGG GGC CCT GAG GTG ACG TTC ATC 4174 Glu Glu Ala Ala Pro Thr Arg Ala Arg Gly Pro Glu Val Thr Phe Ile 2990 2995 3000 3005 TAC CGA GAC CCA GGC TCC TAT CTT GTG ACA GTC ACC GCG TCC AAC AAC 4222 Tyr Arg Asp Pro Gly Ser Tyr Leu Val Thr Val Thr Ala Ser Asn Asn 3010 3015 3020 ATC TCT GCT GCC AAT GAC TCA GCC CTG GTG GAG GTG CAG GAG CCC GTG 4270 Ile Ser Ala Ala Asn Asp Ser Ala Leu Val Glu Val Gln Glu Pro Val 3025 3030 3035 CTG GTC ACC AGC ATC AAG GTC AAT GGC TCC CTT GGG CTG GAG CTG CAG 4318 Leu Val Thr Ser Ile Lys Val Asn Gly Ser Leu Gly Leu Glu Leu Gln 3040 3045 3050 CAG CCG TAC CTG TTC TCT GCT GTG GGC CGT GGG CGC CCC GCC AGC TAC 4366 Gln Pro Tyr Leu Phe Ser Ala Val Gly Arg Gly Arg Pro Ala Ser Tyr 3055 3060 3065 CTG TGG GAT CTG GGG GAC GGT GGG TGG CTC GAG GGT CCG GAG GTC ACC 4414 Leu Trp Asp Leu Gly Asp Gly Gly Trp Leu Glu Gly Pro Glu Val Thr 3070 3075 3080 3085 CAC GCT TAC AAC AGC ACA GGT GAC TTC ACC GTT AGG GTG GCC GGC TGG 4462 His Ala Tyr Asn Ser Thr Gly Asp Phe Thr Val Arg Val Ala Gly Trp 3090 3095 3100 AAT GAG GTG AGC CGC AGC GAG GCC TGG CTC AAT GTG ACG GTG AAG CGG 4510 Asn Glu Val Ser Arg Ser Glu Ala Trp Leu Asn Val Thr Val Lys Arg 3105 3110 3115 CGC GTG CGG GGG CTC GTC GTC AAT GCA AGC CGC ACG GTG GTG CCC CTG 4558 Arg Val Arg Gly Leu Val Val Asn Ala Ser Arg Thr Val Val Pro Leu 3120 3125 3130 AAT GGG AGC GTG AGC TTC AGC ACG TCG CTG GAG GCC GGC AGT GAT GTG 4606 Asn Gly Ser Val Ser Phe Ser Thr Ser Leu Glu Ala Gly Ser Asp Val 3135 3140 3145 CGC TAT TCC TGG GTG CTC TGT GAC CGC TGC ACG CCC ATC CCT GGG GGT 4654 Arg Tyr Ser Trp Val Leu Cys Asp Arg Cys Thr Pro Ile Pro Gly Gly 3150 3155 3160 3165 CCT ACC ATC TCT TAC ACC TTC CGC TCC GTG GGC ACC TTC AAT ATC ATC 4702 Pro Thr Ile Ser Tyr Thr Phe Arg Ser Val Gly Thr Phe Asn Ile Ile 3170 3175 3180 GTC ACG GCT GAG AAC GAG GTG GGC TCC GCC CAG GAC AGC ATC TTC GTC 4750 Val Thr Ala Glu Asn Glu Val Gly Ser Ala Gln Asp Ser Ile Phe Val 3185 3190 3195 TAT GTC CTG CAG CTC ATA GAG GGG CTG CAG GTG GTG GGC GGT GGC CGC 4798 Tyr Val Leu Gln Leu Ile Glu Gly Leu Gln Val Val Gly Gly Gly Arg 3200 3205 3210 TAC TTC CCC ACC AAC CAC ACG GTA CAG CTG CAG GCC GTG GTT AGG GAT 4846 Tyr Phe Pro Thr Asn His Thr Val Gln Leu Gln Ala Val Val Arg Asp 3215 3220 3225 GGC ACC AAC GTC TCC TAC AGC TGG ACT GCC TGG AGG GAC AGG GGC CCG 4894 Gly Thr Asn Val Ser Tyr Ser Trp Thr Ala Trp Arg Asp Arg Gly Pro 3230 3235 3240 3245 GCC CTG GCC GGC AGC GGC AAA GGC TTC TCG CTC ACC GTG CTC GAG GCC 4942 Ala Leu Ala Gly Ser Gly Lys Gly Phe Ser Leu Thr Val Leu Glu Ala 3250 3255 3260 GGC ACC TAC CAT GTG CAG CTG CGG GCC ACC AAC ATG CTG GGC AGC GCC 4990 Gly Thr Tyr His Val Gln Leu Arg Ala Thr Asn Met Leu Gly Ser Ala 3265 3270 3275 TGG GCC GAC TGC ACC ATG GAC TTC GTG GAG CCT GTG GGG TGG CTG ATG 5038 Trp Ala Asp Cys Thr Met Asp Phe Val Glu Pro Val Gly Trp Leu Met 3280 3285 3290 GTG ACC GCC TCC CCG AAC CCA GCT GCC GTC AAC ACA AGC GTC ACC CTC 5086 Val Thr Ala Ser Pro Asn Pro Ala Ala Val Asn Thr Ser Val Thr Leu 3295 3300 3305 AGT GCC GAG CTG GCT GGT GGC AGT GGT GTC GTA TAC ACT TGG TCC TTG 5134 Ser Ala Glu Leu Ala Gly Gly Ser Gly Val Val Tyr Thr Trp Ser Leu 3310 3315 3320 3325 GAG GAG GGG CTG AGC TGG GAG ACC TCC GAG CCA TTT ACC ACC CAT AGC 5182 Glu Glu Gly Leu Ser Trp Glu Thr Ser Glu Pro Phe Thr Thr His Ser 3330 3335 3340 TTC CCC ACA CCC GGC CTG CAC TTG GTC ACC ATG ACG GCA GGG AAC CCG 5230 Phe Pro Thr Pro Gly Leu His Leu Val Thr Met Thr Ala Gly Asn Pro 3345 3350 3355 CTG GGC TCA GCC AAC GCC ACC GTG GAA GTG GAT GTG CAG GTG CCT GTG 5278 Leu Gly Ser Ala Asn Ala Thr Val Glu Val Asp Val Gln Val Pro Val 3360 3365 3370 AGT GGC CTC AGC ATC AGG GCC AGC GAG CCC GGA GGC AGC TTC GTG GCG 5326 Ser Gly Leu Ser Ile Arg Ala Ser Glu Pro Gly Gly Ser Phe Val Ala 3375 3380 3385 GCC GGG TCC TCT GTG CCC TTT TGG GGG CAG CTG GCC ACG GGC ACC AAT 5374 Ala Gly Ser Ser Val Pro Phe Trp Gly Gln Leu Ala Thr Gly Thr Asn 3390 3395 3400 3405 GTG AGC TGG TGC TGG GCT GTG CCC GGC GGC AGC AGC AAG CGT GGC CCT 5422 Val Ser Trp Cys Trp Ala Val Pro Gly Gly Ser Ser Lys Arg Gly Pro 3410 3415 3420 CAT GTC ACC ATG GTC TTC CCG GAT GCT GGC ACC TTC TCC ATC CGG CTC 5470 His Val Thr Met Val Phe Pro Asp Ala Gly Thr Phe Ser Ile Arg Leu 3425 3430 3435 AAT GCC TCC AAC GCA GTC AGC TGG GTC TCA GCC ACG TAC AAC CTC ACG 5518 Asn Ala Ser Asn Ala Val Ser Trp Val Ser Ala Thr Tyr Asn Leu Thr 3440 3445 3450 GCG GAG GAG CCC ATC GTG GGC CTG GTG CTG TGG GCC AGC AGC AAG GTG 5566 Ala Glu Glu Pro Ile Val Gly Leu Val Leu Trp Ala Ser Ser Lys Val 3455 3460 3465 GTG GCG CCC GGG CAG CTG GTC CAT TTT CAG ATC CTG CTG GCT GCC GGC 5614 Val Ala Pro Gly Gln Leu Val His Phe Gln Ile Leu Leu Ala Ala Gly 3470 3475 3480 3485 TCA GCT GTC ACC TTC CGC CTG CAG GTC GGC GGG GCC AAC CCC GAG GTG 5662 Ser Ala Val Thr Phe Arg Leu Gln Val Gly Gly Ala Asn Pro Glu Val 3490 3495 3500 CTC CCC GGG CCC CGT TTC TCC CAC AGC TTC CCC CGC GTC GGA GAC CAC 5710 Leu Pro Gly Pro Arg Phe Ser His Ser Phe Pro Arg Val Gly Asp His 3505 3510 3515 GTG GTG AGC GTG CGG GGC AAA AAC CAC GTG AGC TGG GCC CAG GCG CAG 5758 Val Val Ser Val Arg Gly Lys Asn His Val Ser Trp Ala Gln Ala Gln 3520 3525 3530 GTG CGC ATC GTG GTG CTG GAG GCC GTG AGT GGG CTG CAG ATG CCC AAC 5806 Val Arg Ile Val Val Leu Glu Ala Val Ser Gly Leu Gln Met Pro Asn 3535 3540 3545 TGC TGC GAG CCT GGC ATC GCC ACG GGC ACT GAG AGG AAC TTC ACA GCC 5854 Cys Cys Glu Pro Gly Ile Ala Thr Gly Thr Glu Arg Asn Phe Thr Ala 3550 3555 3560 3565 CGC GTG CAG CGC GGC TCT CGG GTC GCC TAC GCC TGG TAC TTC TCG CTG 5902 Arg Val Gln Arg Gly Ser Arg Val Ala Tyr Ala Trp Tyr Phe Ser Leu 3570 3575 3580 CAG AAG GTC CAG GGC GAC TCG CTG GTC ATC CTG TCG GGC CGC GAC GTC 5950 Gln Lys Val Gln Gly Asp Ser Leu Val Ile Leu Ser Gly Arg Asp Val 3585 3590 3595 ACC TAC ACG CCC GTG GCC GCG GGG CTG TTG GAG ATC CAG GTG CGC GCC 5998 Thr Tyr Thr Pro Val Ala Ala Gly Leu Leu Glu Ile Gln Val Arg Ala 3600 3605 3610 TTC AAC GCC CTG GGC AGT GAG AAC CGC ACG CTG GTG CTG GAG GTT CAG 6046 Phe Asn Ala Leu Gly Ser Glu Asn Arg Thr Leu Val Leu Glu Val Gln 3615 3620 3625 GAC GCC GTC CAG TAT GTG GCC CTG CAG AGC GGC CCC TGC TTC ACC AAC 6094 Asp Ala Val Gln Tyr Val Ala Leu Gln Ser Gly Pro Cys Phe Thr Asn 3630 3635 3640 3645 CGC TCG GCG CAG TTT GAG GCC GCC ACC AGC CCC AGC CCC CGG CGT GTG 6142 Arg Ser Ala Gln Phe Glu Ala Ala Thr Ser Pro Ser Pro Arg Arg Val 3650 3655 3660 GCC TAC CAC TGG GAC TTT GGG GAT GGG TCG CCA GGG CAG GAC ACA GAT 6190 Ala Tyr His Trp Asp Phe Gly Asp Gly Ser Pro Gly Gln Asp Thr Asp 3665 3670 3675 GAG CCC AGG GCC GAG CAC TCC TAC CTG AGG CCT GGG GAC TAC CGC GTG 6238 Glu Pro Arg Ala Glu His Ser Tyr Leu Arg Pro Gly Asp Tyr Arg Val 3680 3685 3690 CAG GTG AAC GCC TCC AAC CTG GTG AGC TTC TTC GTG GCG CAG GCC ACG 6286 Gln Val Asn Ala Ser Asn Leu Val Ser Phe Phe Val Ala Gln Ala Thr 3695 3700 3705 GTG ACC GTC CAG GTG CTG GCC TGC CGG GAG CCG GAG GTG GAC GTG GTC 6334 Val Thr Val Gln Val Leu Ala Cys Arg Glu Pro Glu Val Asp Val Val 3710 3715 3720 3725 CTG CCC CTG CAG GTG CTG ATG CGG CGA TCA CAG CGC AAC TAC TTG GAG 6382 Leu Pro Leu Gln Val Leu Met Arg Arg Ser Gln Arg Asn Tyr Leu Glu 3730 3735 3740 GCC CAC GTT GAC CTG CGC GAC TGC GTC ACC TAC CAG ACT GAG TAC CGC 6430 Ala His Val Asp Leu Arg Asp Cys Val Thr Tyr Gln Thr Glu Tyr Arg 3745 3750 3755 TGG GAG GTG TAT CGC ACC GCC AGC TGC CAG CGG CCG GGG CGC CCA GCG 6478 Trp Glu Val Tyr Arg Thr Ala Ser Cys Gln Arg Pro Gly Arg Pro Ala 3760 3765 3770 CGT GTG GCC CTG CCC GGC GTG GAC GTG AGC CGG CCT CGG CTG GTG CTG 6526 Arg Val Ala Leu Pro Gly Val Asp Val Ser Arg Pro Arg Leu Val Leu 3775 3780 3785 CCG CGG CTG GCG CTG CCT GTG GGG CAC TAC TGC TTT GTG TTT GTC GTG 6574 Pro Arg Leu Ala Leu Pro Val Gly His Tyr Cys Phe Val Phe Val Val 3790 3795 3800 3805 TCA TTT GGG GAC ACG CCA CTG ACA CAG AGC ATC CAG GCC AAT GTG ACG 6622 Ser Phe Gly Asp Thr Pro Leu Thr Gln Ser Ile Gln Ala Asn Val Thr 3810 3815 3820 GTG GCC CCC GAG CGC CTG GTG CCC ATC ATT GAG GGT GGC TCA TAC CGC 6670 Val Ala Pro Glu Arg Leu Val Pro Ile Ile Glu Gly Gly Ser Tyr Arg 3825 3830 3835 GTG TGG TCA GAC ACA CGG GAC CTG GTG CTG GAT GGG AGC GAG TCC TAC 6718 Val Trp Ser Asp Thr Arg Asp Leu Val Leu Asp Gly Ser Glu Ser Tyr 3840 3845 3850 GAC CCC AAC CTG GAG GAC GGC GAC CAG ACG CCG CTC AGT TTC CAC TGG 6766 Asp Pro Asn Leu Glu Asp Gly Asp Gln Thr Pro Leu Ser Phe His Trp 3855 3860 3865 GCC TGT GTG GCT TCG ACA CAG AGG GAG GCT GGC GGG TGT GCG CTG AAC 6814 Ala Cys Val Ala Ser Thr Gln Arg Glu Ala Gly Gly Cys Ala Leu Asn 3870 3875 3880 3885 TTT GGG CCC CGC GGG AGC AGC ACG GTC ACC ATT CCA CGG GAG CGG CTG 6862 Phe Gly Pro Arg Gly Ser Ser Thr Val Thr Ile Pro Arg Glu Arg Leu 3890 3895 3900 GCG GCT GGC GTG GAG TAC ACC TTC AGC CTG ACC GTG TGG AAG GCC GGC 6910 Ala Ala Gly Val Glu Tyr Thr Phe Ser Leu Thr Val Trp Lys Ala Gly 3905 3910 3915 CGC AAG GAG GAG GCC ACC AAC CAG ACG GTG CTG ATC CGG AGT GGC CGG 6958 Arg Lys Glu Glu Ala Thr Asn Gln Thr Val Leu Ile Arg Ser Gly Arg 3920 3925 3930 GTG CCC ATT GTG TCC TTG GAG TGT GTG TCC TGC AAG GCA CAG GCC GTG 7006 Val Pro Ile Val Ser Leu Glu Cys Val Ser Cys Lys Ala Gln Ala Val 3935 3940 3945 TAC GAA GTG AGC CGC AGC TCC TAC GTG TAC TTG GAG GGC CGC TGC CTC 7054 Tyr Glu Val Ser Arg Ser Ser Tyr Val Tyr Leu Glu Gly Arg Cys Leu 3950 3955 3960 3965 AAT TGC AGC AGC GGC TCC AAG CGA GGG CGG TGG GCT GCA CGT ACG TTC 7102 Asn Cys Ser Ser Gly Ser Lys Arg Gly Arg Trp Ala Ala Arg Thr Phe 3970 3975 3980 AGC AAC AAG ACG CTG GTG CTG GAT GAG ACC ACC ACA TCC ACG GGC AGT 7150 Ser Asn Lys Thr Leu Val Leu Asp Glu Thr Thr Thr Ser Thr Gly Ser 3985 3990 3995 GCA GGC ATG CGA CTG GTG CTG CGG CGG GGC GTG CTG CGG GAC GGC GAG 7198 Ala Gly Met Arg Leu Val Leu Arg Arg Gly Val Leu Arg Asp Gly Glu 4000 4005 4010 GGA TAC ACC TTC ACG CTC ACG GTG CTG GGC CGC TCT GGC GAG GAG GAG 7246 Gly Tyr Thr Phe Thr Leu Thr Val Leu Gly Arg Ser Gly Glu Glu Glu 4015 4020 4025 GGC TGC GCC TCC ATC CGC CTG TCC CCC AAC CGC CCG CCG CTG GGG GGC 7294 Gly Cys Ala Ser Ile Arg Leu Ser Pro Asn Arg Pro Pro Leu Gly Gly 4030 4035 4040 4045 TCT TGC CGC CTC TTC CCA CTG GGC GCT GTG CAC GCC CTC ACC ACC AAG 7342 Ser Cys Arg Leu Phe Pro Leu Gly Ala Val His Ala Leu Thr Thr Lys 4050 4055 4060 GTG CAC TTC GAA TGC ACG GGC TGG CAT GAC GCG GAG GAT GCT GGC GCC 7390 Val His Phe Glu Cys Thr Gly Trp His Asp Ala Glu Asp Ala Gly Ala 4065 4070 4075 CCG CTG GTG TAC GCC CTG CTG CTG CGG CGC TGT CGC CAG GGC CAC TGC 7438 Pro Leu Val Tyr Ala Leu Leu Leu Arg Arg Cys Arg Gln Gly His Cys 4080 4085 4090 GAG GAG TTC TGT GTC TAC AAG GGC AGC CTC TCC AGC TAC GGA GCC GTG 7486 Glu Glu Phe Cys Val Tyr Lys Gly Ser Leu Ser Ser Tyr Gly Ala Val 4095 4100 4105 CTG CCC CCG GGT TTC AGG CCA CAC TTC GAG GTG GGC CTG GCC GTG GTG 7534 Leu Pro Pro Gly Phe Arg Pro His Phe Glu Val Gly Leu Ala Val Val 4110 4115 4120 4125 GTG CAG GAC CAG CTG GGA GCC GCT GTG GTC GCC CTC AAC AGG TCT TTG 7582 Val Gln Asp Gln Leu Gly Ala Ala Val Val Ala Leu Asn Arg Ser Leu 4130 4135 4140 GCC ATC ACC CTC CCA GAG CCC AAC GGC AGC GCA ACG GGG CTC ACA GTC 7630 Ala Ile Thr Leu Pro Glu Pro Asn Gly Ser Ala Thr Gly Leu Thr Val 4145 4150 4155 TGG CTG CAC GGG CTC ACC GCT AGT GTG CTC CCA GGG CTG CTG CGG CAG 7678 Trp Leu His Gly Leu Thr Ala Ser Val Leu Pro Gly Leu Leu Arg Gln 4160 4165 4170 GCC GAT CCC CAG CAC GTC ATC GAG TAC TCG TTG GCC CTG GTC ACC GTG 7726 Ala Asp Pro Gln His Val Ile Glu Tyr Ser Leu Ala Leu Val Thr Val 4175 4180 4185 CTG AAC GAG TAC GAG CGG GCC CTG GAC GTG GCG GCA GAG CCC AAG CAC 7774 Leu Asn Glu Tyr Glu Arg Ala Leu Asp Val Ala Ala Glu Pro Lys His 4190 4195 4200 4205 GAG CGG CAG CAC CGA GCC CAG ATA CGC AAG AAC ATC ACG GAG ACT CTG 7822 Glu Arg Gln His Arg Ala Gln Ile Arg Lys Asn Ile Thr Glu Thr Leu 4210 4215 4220 GTG TCC CTG AGG GTC CAC ACT GTG GAT GAC ATC CAG CAG ATC GCT GCT 7870 Val Ser Leu Arg Val His Thr Val Asp Asp Ile Gln Gln Ile Ala Ala 4225 4230 4235 GCG CTG GCC CAG TGC ATG GGG CCC AGC AGG GAG CTC GTA TGC CGC TCG 7918 Ala Leu Ala Gln Cys Met Gly Pro Ser Arg Glu Leu Val Cys Arg Ser 4240 4245 4250 TGC CTG AAG CAG ACG CTG CAC AAG CTG GAG GCC ATG ATG CTC ATC CTG 7966 Cys Leu Lys Gln Thr Leu His Lys Leu Glu Ala Met Met Leu Ile Leu 4255 4260 4265 CAG GCA GAG ACC ACC GCG GGC ACC GTG ACG CCC ACC GCC ATC GGA GAC 8014 Gln Ala Glu Thr Thr Ala Gly Thr Val Thr Pro Thr Ala Ile Gly Asp 4270 4275 4280 4285 AGC ATC CTC AAC ATC ACA GGA GAC CTC ATC CAC CTG GCC AGC TCG GAC 8062 Ser Ile Leu Asn Ile Thr Gly Asp Leu Ile His Leu Ala Ser Ser Asp 4290 4295 4300 GTG CGG GCA CCA CAG CCC TCA GAG CTG GGA GCC GAG TCA CCA TCT CGG 8110 Val Arg Ala Pro Gln Pro Ser Glu Leu Gly Ala Glu Ser Pro Ser Arg 4305 4310 4315 ATG GTG GCG TCC CAG GCC TAC AAC CTG ACC TCT GCC CTC ATG CGC ATC 8158 Met Val Ala Ser Gln Ala Tyr Asn Leu Thr Ser Ala Leu Met Arg Ile 4320 4325 4330 CTC ATG CGC TCC CGC GTG CTC AAC GAG GAG CCC CTG ACG CTG GCG GGC 8206 Leu Met Arg Ser Arg Val Leu Asn Glu Glu Pro Leu Thr Leu Ala Gly 4335 4340 4345 GAG GAG ATC GTG GCC CAG GGC AAG CGC TCG GAC CCG CGG AGC CTG CTG 8254 Glu Glu Ile Val Ala Gln Gly Lys Arg Ser Asp Pro Arg Ser Leu Leu 4350 4355 4360 4365 TGC TAT GGC GGC GCC CCA GGG CCT GGC TGC CAC TTC TCC ATC CCC GAG 8302 Cys Tyr Gly Gly Ala Pro Gly Pro Gly Cys His Phe Ser Ile Pro Glu 4370 4375 4380 GCT TTC AGC GGG GCC CTG GCC AAC CTC AGT GAC GTG GTG CAG CTC ATC 8350 Ala Phe Ser Gly Ala Leu Ala Asn Leu Ser Asp Val Val Gln Leu Ile 4385 4390 4395 TTT CTG GTG GAC TCC AAT CCC TTT CCC TTT GGC TAT ATC AGC AAC TAC 8398 Phe Leu Val Asp Ser Asn Pro Phe Pro Phe Gly Tyr Ile Ser Asn Tyr 4400 4405 4410 ACC GTC TCC ACC AAG GTG GCC TCG ATG GCA TTC CAG ACA CAG GCC GGC 8446 Thr Val Ser Thr Lys Val Ala Ser Met Ala Phe Gln Thr Gln Ala Gly 4415 4420 4425 GCC CAG ATC CCC ATC GAG CGG CTG GCC TCA GAG CGC GCC ATC ACC GTG 8494 Ala Gln Ile Pro Ile Glu Arg Leu Ala Ser Glu Arg Ala Ile Thr Val 4430 4435 4440 4445 AAG GTG CCC AAC AAC TCG GAC TGG GCT GCC CGG GGC CAC CGC AGC TCC 8542 Lys Val Pro Asn Asn Ser Asp Trp Ala Ala Arg Gly His Arg Ser Ser 4450 4455 4460 GCC AAC TCC GCC AAC TCC GTT GTG GTC CAG CCC CAG GCC TCC GTC GGT 8590 Ala Asn Ser Ala Asn Ser Val Val Val Gln Pro Gln Ala Ser Val Gly 4465 4470 4475 GCT GTG GTC ACC CTG GAC AGC AGC AAC CCT GCG GCC GGG CTG CAT CTG 8638 Ala Val Val Thr Leu Asp Ser Ser Asn Pro Ala Ala Gly Leu His Leu 4480 4485 4490 CAG CTC AAC TAT ACG CTG CTG GAC GGC CAC TAC CTG TCT GAG GAA CCT 8686 Gln Leu Asn Tyr Thr Leu Leu Asp Gly His Tyr Leu Ser Glu Glu Pro 4495 4500 4505 GAG CCC TAC CTG GCA GTC TAC CTA CAC TCG GAG CCC CGG CCC AAT GAG 8734 Glu Pro Tyr Leu Ala Val Tyr Leu His Ser Glu Pro Arg Pro Asn Glu 4510 4515 4520 4525 CAC AAC TGC TCG GCT AGC AGG AGG ATC CGC CCA GAG TCA CTC CAG GGT 8782 His Asn Cys Ser Ala Ser Arg Arg Ile Arg Pro Glu Ser Leu Gln Gly 4530 4535 4540 GCT GAC CAC CGG CCC TAC ACC TTC TTC ATT TCC CCG GGG AGC AGA GAC 8830 Ala Asp His Arg Pro Tyr Thr Phe Phe Ile Ser Pro Gly Ser Arg Asp 4545 4550 4555 CCA GCG GGG AGT TAC CAT CTG AAC CTC TCC AGC CAC TTC CGC TGG TCG 8878 Pro Ala Gly Ser Tyr His Leu Asn Leu Ser Ser His Phe Arg Trp Ser 4560 4565 4570 GCG CTG CAG GTG TCC GTG GGC CTG TAC ACG TCC CTG TGC CAG TAC TTC 8926 Ala Leu Gln Val Ser Val Gly Leu Tyr Thr Ser Leu Cys Gln Tyr Phe 4575 4580 4585 AGC GAG GAG GAC ATG GTG TGG CGG ACA GAG GGG CTG CTG CCC CTG GAG 8974 Ser Glu Glu Asp Met Val Trp Arg Thr Glu Gly Leu Leu Pro Leu Glu 4590 4595 4600 4605 GAG ACC TCG CCC CGC CAG GCC GTC TGC CTC ACC CGC CAC CTC ACC GCC 9022 Glu Thr Ser Pro Arg Gln Ala Val Cys Leu Thr Arg His Leu Thr Ala 4610 4615 4620 TTC GGC GCC AGC CTC TTC GTG CCC CCA AGC CAT GTC CGC TTT GTG TTT 9070 Phe Gly Ala Ser Leu Phe Val Pro Pro Ser His Val Arg Phe Val Phe 4625 4630 4635 CCT GAG CCG ACA GCG GAT GTA AAC TAC ATC GTC ATG CTG ACA TGT GCT 9118 Pro Glu Pro Thr Ala Asp Val Asn Tyr Ile Val Met Leu Thr Cys Ala 4640 4645 4650 GTG TGC CTG GTG ACC TAC ATG GTC ATG GCC GCC ATC CTG CAC AAG CTG 9166 Val Cys Leu Val Thr Tyr Met Val Met Ala Ala Ile Leu His Lys Leu 4655 4660 4665 GAC CAG TTG GAT GCC AGC CGG GGC CGC GCC ATC CCT TTC TGT GGG CAG 9214 Asp Gln Leu Asp Ala Ser Arg Gly Arg Ala Ile Pro Phe Cys Gly Gln 4670 4675 4680 4685 CGG GGC CGC TTC AAG TAC GAG ATC CTC GTC AAG ACA GGC TGG GGC CGG 9262 Arg Gly Arg Phe Lys Tyr Glu Ile Leu Val Lys Thr Gly Trp Gly Arg 4690 4695 4700 GGC TCA GGT ACC ACG GCC CAC GTG GGC ATC ATG CTG TAT GGG GTG GAC 9310 Gly Ser Gly Thr Thr Ala His Val Gly Ile Met Leu Tyr Gly Val Asp 4705 4710 4715 AGC CGG AGC GGC CAC CGG CAC CTG GAC GGC GAC AGA GCC TTC CAC CGC 9358 Ser Arg Ser Gly His Arg His Leu Asp Gly Asp Arg Ala Phe His Arg 4720 4725 4730 AAC AGC CTG GAC ATC TTC CGG ATC GCC ACC CCG CAC AGC CTG GGT AGC 9406 Asn Ser Leu Asp Ile Phe Arg Ile Ala Thr Pro His Ser Leu Gly Ser 4735 4740 4745 GTG TGG AAG ATC CGA GTG TGG CAC GAC AAC AAA GGG CTC AGC CCT GCC 9454 Val Trp Lys Ile Arg Val Trp His Asp Asn Lys Gly Leu Ser Pro Ala 4750 4755 4760 4765 TGG TTC CTG CAG CAC GTC ATC GTC AGG GAC CTG CAG ACG GCA CGC AGC 9502 Trp Phe Leu Gln His Val Ile Val Arg Asp Leu Gln Thr Ala Arg Ser 4770 4775 4780 GCC TTC TTC CTG GTC AAT GAC TGG CTT TCG GTG GAG ACG GAG GCC AAC 9550 Ala Phe Phe Leu Val Asn Asp Trp Leu Ser Val Glu Thr Glu Ala Asn 4785 4790 4795 GGG GGC CTG GTG GAG AAG GAG GTG CTG GCC GCG AGC GAC GCA GCC CTT 9598 Gly Gly Leu Val Glu Lys Glu Val Leu Ala Ala Ser Asp Ala Ala Leu 4800 4805 4810 TTG CGC TTC CGG CGC CTG CTG GTG GCT GAG CTG CAG CGT GGC TTC TTT 9646 Leu Arg Phe Arg Arg Leu Leu Val Ala Glu Leu Gln Arg Gly Phe Phe 4815 4820 4825 GAC AAG CAC ATC TGG CTC TCC ATA TGG GAC CGG CCG CCT CGT AGC CGT 9694 Asp Lys His Ile Trp Leu Ser Ile Trp Asp Arg Pro Pro Arg Ser Arg 4830 4835 4840 4845 TTC ACT CGC ATC CAG AGG GCC ACC TGC TGC GTT CTC CTC ATC TGC CTC 9742 Phe Thr Arg Ile Gln Arg Ala Thr Cys Cys Val Leu Leu Ile Cys Leu 4850 4855 4860 TTC CTG GGC GCC AAC GCC GTG TGG TAC GGG GCT GTT GGC GAC TCT GCC 9790 Phe Leu Gly Ala Asn Ala Val Trp Tyr Gly Ala Val Gly Asp Ser Ala 4865 4870 4875 TAC AGC ACG GGG CAT GTG TCC AGG CTG AGC CCG CTG AGC GTC GAC ACA 9838 Tyr Ser Thr Gly His Val Ser Arg Leu Ser Pro Leu Ser Val Asp Thr 4880 4885 4890 GTC GCT GTT GGC CTG GTG TCC AGC GTG GTT GTC TAT CCC GTC TAC CTG 9886 Val Ala Val Gly Leu Val Ser Ser Val Val Val Tyr Pro Val Tyr Leu 4895 4900 4905 GCC ATC CTT TTT CTC TTC CGG ATG TCC CGG AGC AAG GTG GCT GGG AGC 9934 Ala Ile Leu Phe Leu Phe Arg Met Ser Arg Ser Lys Val Ala Gly Ser 4910 4915 4920 4925 CCG AGC CCC ACA CCT GCC GGG CAG CAG GTG CTG GAC ATC GAC AGC TGC 9982 Pro Ser Pro Thr Pro Ala Gly Gln Gln Val Leu Asp Ile Asp Ser Cys 4930 4935 4940 CTG GAC TCG TCC GTG CTG GAC AGC TCC TTC CTC ACG TTC TCA GGC CTC 10030 Leu Asp Ser Ser Val Leu Asp Ser Ser Phe Leu Thr Phe Ser Gly Leu 4945 4950 4955 CAC GCT GAG GCC TTT GTT GGA CAG ATG AAG AGT GAC TTG TTT CTG GAT 10078 His Ala Glu Ala Phe Val Gly Gln Met Lys Ser Asp Leu Phe Leu Asp 4960 4965 4970 GAT TCT AAG AGT CTG GTG TGC TGG CCC TCC GGC GAG GGA ACG CTC AGT 10126 Asp Ser Lys Ser Leu Val Cys Trp Pro Ser Gly Glu Gly Thr Leu Ser 4975 4980 4985 TGG CCG GAC CTG CTC AGT GAC CCG TCC ATT GTG GGT AGC AAT CTG CGG 10174 Trp Pro Asp Leu Leu Ser Asp Pro Ser Ile Val Gly Ser Asn Leu Arg 4990 4995 5000 5005 CAG CTG GCA CGG GGC CAG GCG GGC CAT GGG CTG GGC CCA GAG GAG GAC 10222 Gln Leu Ala Arg Gly Gln Ala Gly His Gly Leu Gly Pro Glu Glu Asp 5010 5015 5020 GGC TTC TCC CTG GCC AGC CCC TAC TCG CCT GCC AAA TCC TTC TCA GCA 10270 Gly Phe Ser Leu Ala Ser Pro Tyr Ser Pro Ala Lys Ser Phe Ser Ala 5025 5030 5035 TCA GAT GAA GAC CTG ATC CAG CAG GTC CTT GCC GAG GGG GTC AGC AGC 10318 Ser Asp Glu Asp Leu Ile Gln Gln Val Leu Ala Glu Gly Val Ser Ser 5040 5045 5050 CCA GCC CCT ACC CAA GAC ACC CAC ATG GAA ACG GAC CTG CTC AGC AGC 10366 Pro Ala Pro Thr Gln Asp Thr His Met Glu Thr Asp Leu Leu Ser Ser 5055 5060 5065 CTG TCC AGC ACT CCT GGG GAG AAG ACA GAG ACG CTG GCG CTG CAG AGG 10414 Leu Ser Ser Thr Pro Gly Glu Lys Thr Glu Thr Leu Ala Leu Gln Arg 5070 5075 5080 5085 CTG GGG GAG CTG GGG CCA CCC AGC CCA GGC CTG AAC TGG GAA CAG CCC 10462 Leu Gly Glu Leu Gly Pro Pro Ser Pro Gly Leu Asn Trp Glu Gln Pro 5090 5095 5100 CAG GCA GCG AGG CTG TCC AGG ACA GGA CTG GTG GAG GGT CTG CGG AAG 10510 Gln Ala Ala Arg Leu Ser Arg Thr Gly Leu Val Glu Gly Leu Arg Lys 5105 5110 5115 CGC CTG CTG CCG GCC TGG TGT GCC TCC CTG GCC CAC GGG CTC AGC CTG 10558 Arg Leu Leu Pro Ala Trp Cys Ala Ser Leu Ala His Gly Leu Ser Leu 5120 5125 5130 CTC CTG GTG GCT GTG GCT GTG GCT GTC TCA GGG TGG GTG GGT GCG AGC 10606 Leu Leu Val Ala Val Ala Val Ala Val Ser Gly Trp Val Gly Ala Ser 5135 5140 5145 TTC CCC CCG GGC GTG AGT GTT GCG TGG CTC CTG TCC AGC AGC GCC AGC 10654 Phe Pro Pro Gly Val Ser Val Ala Trp Leu Leu Ser Ser Ser Ala Ser 5150 5155 5160 5165 TTC CTG GCC TCA TTC CTC GGC TGG GAG CCA CTG AAG GTC TTG CTG GAA 10702 Phe Leu Ala Ser Phe Leu Gly Trp Glu Pro Leu Lys Val Leu Leu Glu 5170 5175 5180 GCC CTG TAC TTC TCA CTG GTG GCC AAG CGG CTG CAC CCG GAT GAA GAT 10750 Ala Leu Tyr Phe Ser Leu Val Ala Lys Arg Leu His Pro Asp Glu Asp 5185 5190 5195 GAC ACC CTG GTA GAG AGC CCG GCT GTG ACG CCT GTG AGC GCA CGT GTG 10798 Asp Thr Leu Val Glu Ser Pro Ala Val Thr Pro Val Ser Ala Arg Val 5200 5205 5210 CCC CGC GTA CGG CCA CCC CAC GGC TTT GCA CTC TTC CTG GCC AAG GAA 10846 Pro Arg Val Arg Pro Pro His Gly Phe Ala Leu Phe Leu Ala Lys Glu 5215 5220 5225 GAA GCC CGC AAG GTC AAG AGG CTA CAT GGC ATG CTG CGG AGC CTC CTG 10894 Glu Ala Arg Lys Val Lys Arg Leu His Gly Met Leu Arg Ser Leu Leu 5230 5235 5240 5245 GTG TAC ATG CTT TTT CTG CTG GTG ACC CTG CTG GCC AGC TAT GGG GAT 10942 Val Tyr Met Leu Phe Leu Leu Val Thr Leu Leu Ala Ser Tyr Gly Asp 5250 5255 5260 GCC TCA TGC CAT GGG CAC GCC TAC CGT CTG CAA AGC GCC ATC AAG CAG 10990 Ala Ser Cys His Gly His Ala Tyr Arg Leu Gln Ser Ala Ile Lys Gln 5265 5270 5275 GAG CTG CAC AGC CGG GCC TTC CTG GCC ATC ACG CGG TCT GAG GAG CTC 11038 Glu Leu His Ser Arg Ala Phe Leu Ala Ile Thr Arg Ser Glu Glu Leu 5280 5285 5290 TGG CCA TGG ATG GCC CAC GTG CTG CTG CCC TAC GTC CAC GGG AAC CAG 11086 Trp Pro Trp Met Ala His Val Leu Leu Pro Tyr Val His Gly Asn Gln 5295 5300 5305 TCC AGC CCA GAG CTG GGG CCC CCA CGG CTG CGG CAG GTG CGG CTG CAG 11134 Ser Ser Pro Glu Leu Gly Pro Pro Arg Leu Arg Gln Val Arg Leu Gln 5310 5315 5320 5325 GAA GCA CTC TAC CCA GAC CCT CCC GGC CCC AGG GTC CAC ACG TGC TCG 11182 Glu Ala Leu Tyr Pro Asp Pro Pro Gly Pro Arg Val His Thr Cys Ser 5330 5335 5340 GCC GCA GGA GGC TTC AGC ACC AGC GAT TAC GAC GTT GGC TGG GAG AGT 11230 Ala Ala Gly Gly Phe Ser Thr Ser Asp Tyr Asp Val Gly Trp Glu Ser 5345 5350 5355 CCT CAC AAT GGC TCG GGG ACG TGG GCC TAT TCA GCG CCG GAT CTG CTG 11278 Pro His Asn Gly Ser Gly Thr Trp Ala Tyr Ser Ala Pro Asp Leu Leu 5360 5365 5370 GGG GCA TGG TCC TGG GGC TCC TGT GCC GTG TAT GAC AGC GGG GGC TAC 11326 Gly Ala Trp Ser Trp Gly Ser Cys Ala Val Tyr Asp Ser Gly Gly Tyr 5375 5380 5385 GTG CAG GAG CTG GGC CTG AGC CTG GAG GAG AGC CGC GAC CGG CTG CGC 11374 Val Gln Glu Leu Gly Leu Ser Leu Glu Glu Ser Arg Asp Arg Leu Arg 5390 5395 5400 5405 TTC CTG CAG CTG CAC AAC TGG CTG GAC AAC AGG AGC CGC GCT GTG TTC 11422 Phe Leu Gln Leu His Asn Trp Leu Asp Asn Arg Ser Arg Ala Val Phe 5410 5415 5420 CTG GAG CTC ACG CGC TAC AGC CCG GCC GTG GGG CTG CAC GCC GCC GTC 11470 Leu Glu Leu Thr Arg Tyr Ser Pro Ala Val Gly Leu His Ala Ala Val 5425 5430 5435 ACG CTG CGC CTC GAG TTC CCG GCG GCC GGC CGC GCC CTG GCC GCC CTC 11518 Thr Leu Arg Leu Glu Phe Pro Ala Ala Gly Arg Ala Leu Ala Ala Leu 5440 5445 5450 AGC GTC CGC CCC TTT GCG CTG CGC CGC CTC AGC GCG GGC CTC TCG CTG 11566 Ser Val Arg Pro Phe Ala Leu Arg Arg Leu Ser Ala Gly Leu Ser Leu 5455 5460 5465 CCT CTG CTC ACC TCG GTG TGC CTG CTG CTG TTC GCC GTG CAC TTC GCC 11614 Pro Leu Leu Thr Ser Val Cys Leu Leu Leu Phe Ala Val His Phe Ala 5470 5475 5480 5485 GTG GCC GAG GCC CGT ACT TGG CAC AGG GAA GGG CGC TGG CGC GTG CTG 11662 Val Ala Glu Ala Arg Thr Trp His Arg Glu Gly Arg Trp Arg Val Leu 5490 5495 5500 CGG CTC GGA GCC TGG GCG CGG TGG CTG CTG GTG GCG CTG ACG GCG GCC 11710 Arg Leu Gly Ala Trp Ala Arg Trp Leu Leu Val Ala Leu Thr Ala Ala 5505 5510 5515 ACG GCA CTG GTA CGC CTC GCC CAG CTG GGT GCC GCT GAC CGC CAG TGG 11758 Thr Ala Leu Val Arg Leu Ala Gln Leu Gly Ala Ala Asp Arg Gln Trp 5520 5525 5530 ACC CGT TTC GTG CGC GGC CGC CCG CGC CGC TTC ACT AGC TTC GAC CAG 11806 Thr Arg Phe Val Arg Gly Arg Pro Arg Arg Phe Thr Ser Phe Asp Gln 5535 5540 5545 GTG GCG CAC GTG AGC TCC GCA GCC CGT GGC CTG GCG GCC TCG CTG CTC 11854 Val Ala His Val Ser Ser Ala Ala Arg Gly Leu Ala Ala Ser Leu Leu 5550 5555 5560 5565 TTC CTG CTT TTG GTC AAG GCT GCC CAG CAC GTA CGC TTC GTG CGC CAG 11902 Phe Leu Leu Leu Val Lys Ala Ala Gln His Val Arg Phe Val Arg Gln 5570 5575 5580 TGG TCC GTC TTT GGC AAG ACA TTA TGC CGA GCT CTG CCA GAG CTC CTG 11950 Trp Ser Val Phe Gly Lys Thr Leu Cys Arg Ala Leu Pro Glu Leu Leu 5585 5590 5595 GGG GTC ACC TTG GGC CTG GTG GTG CTC GGG GTA GCC TAC GCC CAG CTG 11998 Gly Val Thr Leu Gly Leu Val Val Leu Gly Val Ala Tyr Ala Gln Leu 5600 5605 5610 GCC ATC CTG CTC GTG TCT TCC TGT GTG GAC TCC CTC TGG AGC GTG GCC 12046 Ala Ile Leu Leu Val Ser Ser Cys Val Asp Ser Leu Trp Ser Val Ala 5615 5620 5625 CAG GCC CTG TTG GTG CTG TGC CCT GGG ACT GGG CTC TCT ACC CTG TGT 12094 Gln Ala Leu Leu Val Leu Cys Pro Gly Thr Gly Leu Ser Thr Leu Cys 5630 5635 5640 5645 CCT GCC GAG TCC TGG CAC CTG TCA CCC CTG CTG TGT GTG GGG CTC TGG 12142 Pro Ala Glu Ser Trp His Leu Ser Pro Leu Leu Cys Val Gly Leu Trp 5650 5655 5660 GCA CTG CGG CTG TGG GGC GCC CTA CGG CTG GGG GCT GTT ATT CTC CGC 12190 Ala Leu Arg Leu Trp Gly Ala Leu Arg Leu Gly Ala Val Ile Leu Arg 5665 5670 5675 TGG CGC TAC CAC GCC TTG CGT GGA GAG CTG TAC CGG CCG GCC TGG GAG 12238 Trp Arg Tyr His Ala Leu Arg Gly Glu Leu Tyr Arg Pro Ala Trp Glu 5680 5685 5690 CCC CAG GAC TAC GAG ATG GTG GAG TTG TTC CTG CGC AGG CTG CGC CTC 12286 Pro Gln Asp Tyr Glu Met Val Glu Leu Phe Leu Arg Arg Leu Arg Leu 5695 5700 5705 TGG ATG GGC CTC AGC AAG GTC AAG GAG TTC CGC CAC AAA GTC CGC TTT 12334 Trp Met Gly Leu Ser Lys Val Lys Glu Phe Arg His Lys Val Arg Phe 5710 5715 5720 5725 GAA GGG ATG GAG CCG CTG CCC TCT CGC TCC TCC AGG GGC TCC AAG GTA 12382 Glu Gly Met Glu Pro Leu Pro Ser Arg Ser Ser Arg Gly Ser Lys Val 5730 5735 5740 TCC CCG GAT GTG CCC CCA CCC AGC GCT GGC TCC GAT GCC TCG CAC CCC 12430 Ser Pro Asp Val Pro Pro Pro Ser Ala Gly Ser Asp Ala Ser His Pro 5745 5750 5755 TCC ACC TCC TCC AGC CAG CTG GAT GGG CTG AGC GTG AGC CTG GGC CGG 12478 Ser Thr Ser Ser Ser Gln Leu Asp Gly Leu Ser Val Ser Leu Gly Arg 5760 5765 5770 CTG GGG ACA AGG TGT GAG CCT GAG CCC TCC CGC CTC CAA GCC GTG TTC 12526 Leu Gly Thr Arg Cys Glu Pro Glu Pro Ser Arg Leu Gln Ala Val Phe 5775 5780 5785 GAG GCC CTG CTC ACC CAG TTT GAC CGA CTC AAC CAG GCC ACA GAG GAC 12574 Glu Ala Leu Leu Thr Gln Phe Asp Arg Leu Asn Gln Ala Thr Glu Asp 5790 5795 5800 5805 GTC TAC CAG CTG GAG CAG CAG CTG CAC AGC CTG CAA GGC CGC AGG AGC 12622 Val Tyr Gln Leu Glu Gln Gln Leu His Ser Leu Gln Gly Arg Arg Ser 5810 5815 5820 AGC CGG GCG CCC GCC GGA TCT TCC CGT GGC CCA TCC CCG GGC CTG CGG 12670 Ser Arg Ala Pro Ala Gly Ser Ser Arg Gly Pro Ser Pro Gly Leu Arg 5825 5830 5835 CCA GCA CTG CCC AGC CGC CTT GCC CGG GCC AGT CGG GGT GTG GAC CTG 12718 Pro Ala Leu Pro Ser Arg Leu Ala Arg Ala Ser Arg Gly Val Asp Leu 5840 5845 5850 GCC ACT GGC CCC AGC AGG ACA CCT TCG GGC CAA GAA CAA GGT CCA CCC 12766 Ala Thr Gly Pro Ser Arg Thr Pro Ser Gly Gln Glu Gln Gly Pro Pro 5855 5860 5865 CAG CAG CAC TTA GTC CTC CTT CCT GGC GGG GGT GGG CCG TGG AGT CGG 12814 Gln Gln His Leu Val Leu Leu Pro Gly Gly Gly Gly Pro Trp Ser Arg 5870 5875 5880 5885 AGT GGA CAC CGC TCA GTA TTA CTT TCT GCC GCT GTC AAG GCC GAG GGC 12862 Ser Gly His Arg Ser Val Leu Leu Ser Ala Ala Val Lys Ala Glu Gly 5890 5895 5900 CAG GCA GAA TGG CTG CAC GTA GGT TCC CCA GAG AGC AGG CAG GGG CAT 12910 Gln Ala Glu Trp Leu His Val Gly Ser Pro Glu Ser Arg Gln Gly His 5905 5910 5915 CTG TCT GTC TGT GGG CTT CAG CAC TTT AAA GAG GCT GTG TGG CCA ACC 12958 Leu Ser Val Cys Gly Leu Gln His Phe Lys Glu Ala Val Trp Pro Thr 5920 5925 5930 AGG ACC CAG GGT CCC CTC CCC AGC TCC CTT GGG AAG GAC ACA GCA GTA 13006 Arg Thr Gln Gly Pro Leu Pro Ser Ser Leu Gly Lys Asp Thr Ala Val 5935 5940 5945 TTG GAC GGT TTC TAGCCTCTGA GATGCTAATT TATTTCCCCG AGTCCTCAGG 13058 Leu Asp Gly Phe 5950 TACAGCGGGC TGTGCCCGGC CCCACCCCCT GGGCAGATGT CCCCCACTGC TAAGGCTGCT 13118 GGCTTCAGGG AGGGTTAGCC TGCACCGCCG CCACCCTGCC CCTAAGTTAT TACCTCTCCA 13178 GTTCCTACCG TACTCCCTGC ACCGTCTCAC TGTGTGTCTC GTGTCAGTAA TTTATATGGT 13238 GTTAAAATGT GTATATTTTT GTATGTCACT ATTTTCACTA GGGCTGAGGG GCCTGCGCCC 13298 AGAGCTGGCC TCCCCCAACA CCTGCTGCGC TTGGTAGGTG TGGTGGCGTT ATGGCAGCCC 13358 GGCTGCTGCT TGGATGCGAG CTTGGCCTTG GGCCGGTGCT GGGGGCACAG CTGTCTGCCA 13418 GGCACTCTCA TCACCCCAGA GGCCTTGTCA TCCTCCCTTG CCCCAGGCCA GGTAGCAAGA 13478 GAGCAGCGCC CAGGCCTGCT GGCATCAGGT CTGGGCAAGT AGCAGGACTA GGCATGTCAG 13538 AGGACCCCAG GGTGGTTAGA GGAAAAGACT CCTCCTGGGG GCTGGCTCCC AGGGTGGAGG 13598 AAGGTGACTG TGTGTGTGTG TGTGTGCGCG CGCGACGCGC GAGTGTGCTG TATGGCCCAG 13658 GCAGCCTCAA GGCCCTCGGA GCTGGCTGTG CCTGCTTCTG TGTACCACTT CTGTGGGCAT 13718 GGCCGCTTCT AGAGCCTCGA CACCCCCCCA ACCCCCGCAC CAAGCAGACA AAGTCAATAA 13778 AAGAGCTGTC TGACTGCAAA AAAAAAAAA 13807 4339 amino acids amino acid linear protein 6 Gly Ala Ala Cys Arg Val Asn Cys Ser Gly Arg Gly Leu Arg Thr Leu 1 5 10 15 Gly Pro Ala Leu Arg Ile Pro Ala Asp Ala Thr Ala Leu Asp Val Ser 20 25 30 His Asn Leu Leu Arg Ala Leu Asp Val Gly Leu Leu Ala Asn Leu Ser 35 40 45 Ala Leu Ala Glu Leu Asp Ile Ser Asn Asn Lys Ile Ser Thr Leu Glu 50 55 60 Glu Gly Ile Phe Ala Asn Leu Phe Asn Leu Ser Glu Ile Asn Leu Ser 65 70 75 80 Gly Asn Pro Phe Glu Cys Asp Cys Gly Leu Ala Trp Leu Pro Arg Trp 85 90 95 Ala Glu Glu Gln Gln Val Arg Val Val Gln Pro Glu Ala Ala Thr Cys 100 105 110 Ala Gly Pro Gly Ser Leu Ala Gly Gln Pro Leu Leu Gly Ile Pro Leu 115 120 125 Leu Asp Ser Gly Cys Gly Glu Glu Tyr Val Ala Cys Leu Pro Asp Asn 130 135 140 Ser Ser Gly Thr Val Ala Ala Val Ser Phe Ser Ala Ala His Glu Gly 145 150 155 160 Leu Leu Gln Pro Glu Ala Cys Ser Ala Phe Cys Phe Ser Thr Gly Gln 165 170 175 Gly Leu Ala Ala Leu Ser Glu Gln Gly Trp Cys Leu Cys Gly Ala Ala 180 185 190 Gln Pro Ser Ser Ala Ser Phe Ala Cys Leu Ser Leu Cys Ser Gly Pro 195 200 205 Pro Pro Pro Pro Ala Pro Thr Cys Arg Gly Pro Thr Leu Leu Gln His 210 215 220 Val Phe Pro Ala Ser Pro Gly Ala Thr Leu Val Gly Pro His Gly Pro 225 230 235 240 Leu Ala Ser Gly Gln Leu Ala Ala Phe His Ile Ala Ala Pro Leu Pro 245 250 255 Val Thr Ala Thr Arg Trp Asp Phe Gly Asp Gly Ser Ala Glu Val Asp 260 265 270 Ala Ala Gly Pro Ala Ala Ser His Arg Tyr Val Leu Pro Gly Arg Tyr 275 280 285 His Val Thr Ala Val Leu Ala Leu Gly Ala Gly Ser Ala Leu Leu Gly 290 295 300 Thr Asp Val Gln Val Glu Ala Ala Pro Ala Ala Leu Glu Leu Val Cys 305 310 315 320 Pro Ser Ser Val Gln Ser Asp Glu Ser Leu Asp Leu Ser Ile Gln Asn 325 330 335 Arg Gly Gly Ser Gly Leu Glu Ala Ala Tyr Ser Ile Val Ala Leu Gly 340 345 350 Glu Glu Pro Ala Arg Ala Val His Pro Leu Cys Pro Ser Asp Thr Glu 355 360 365 Ile Phe Pro Gly Asn Gly His Cys Tyr Arg Leu Val Val Glu Lys Ala 370 375 380 Ala Trp Leu Gln Ala Gln Glu Gln Cys Gln Ala Trp Ala Gly Ala Ala 385 390 395 400 Leu Ala Met Val Asp Ser Pro Ala Val Gln Arg Phe Leu Val Ser Arg 405 410 415 Val Thr Arg Ser Leu Asp Val Trp Ile Gly Phe Ser Thr Val Gln Gly 420 425 430 Val Glu Val Gly Pro Ala Pro Gln Gly Glu Ala Phe Ser Leu Glu Ser 435 440 445 Cys Gln Asn Trp Leu Pro Gly Glu Pro His Pro Ala Thr Ala Glu His 450 455 460 Cys Val Arg Leu Gly Pro Thr Gly Trp Cys Asn Thr Asp Leu Cys Ser 465 470 475 480 Ala Pro His Ser Tyr Val Cys Glu Leu Gln Pro Gly Gly Pro Val Gln 485 490 495 Asp Ala Glu Asn Leu Leu Val Gly Ala Pro Ser Gly Asp Leu Gln Gly 500 505 510 Pro Leu Thr Pro Leu Ala Gln Gln Asp Gly Leu Ser Ala Pro His Glu 515 520 525 Pro Val Glu Val Met Val Phe Pro Gly Leu Arg Leu Ser Arg Glu Ala 530 535 540 Phe Leu Thr Thr Ala Glu Phe Gly Thr Gln Glu Leu Arg Arg Pro Ala 545 550 555 560 Gln Leu Arg Leu Gln Val Tyr Arg Leu Leu Ser Thr Ala Gly Thr Pro 565 570 575 Glu Asn Gly Ser Glu Pro Glu Ser Arg Ser Pro Asp Asn Arg Thr Gln 580 585 590 Leu Ala Pro Ala Cys Met Pro Gly Gly Arg Trp Cys Pro Gly Ala Asn 595 600 605 Ile Cys Leu Pro Leu Asp Ala Ser Cys His Pro Gln Ala Cys Ala Asn 610 615 620 Gly Cys Thr Ser Gly Pro Gly Leu Pro Gly Ala Pro Tyr Ala Leu Trp 625 630 635 640 Arg Glu Phe Leu Phe Ser Val Ala Ala Gly Pro Pro Ala Gln Tyr Ser 645 650 655 Val Thr Leu His Gly Gln Asp Val Leu Met Leu Pro Gly Asp Leu Val 660 665 670 Gly Leu Gln His Asp Ala Gly Pro Gly Ala Leu Leu His Cys Ser Pro 675 680 685 Ala Pro Gly His Pro Gly Pro Gln Ala Pro Tyr Leu Ser Ala Asn Ala 690 695 700 Ser Ser Trp Leu Pro His Leu Pro Ala Gln Leu Glu Gly Thr Trp Ala 705 710 715 720 Cys Pro Ala Cys Ala Leu Arg Leu Leu Ala Ala Thr Glu Gln Leu Thr 725 730 735 Val Leu Leu Gly Leu Arg Pro Asn Pro Gly Leu Arg Met Pro Gly Arg 740 745 750 Tyr Glu Val Arg Ala Glu Val Gly Asn Gly Val Ser Arg His Asn Leu 755 760 765 Ser Cys Ser Phe Asp Val Val Ser Pro Val Ala Gly Leu Arg Val Ile 770 775 780 Tyr Pro Ala Pro Arg Asp Gly Arg Leu Tyr Val Pro Thr Asn Gly Ser 785 790 795 800 Ala Leu Val Leu Gln Val Asp Ser Gly Ala Asn Ala Thr Ala Thr Ala 805 810 815 Arg Trp Pro Gly Gly Ser Val Ser Ala Arg Phe Glu Asn Val Cys Pro 820 825 830 Ala Leu Val Ala Thr Phe Val Pro Gly Cys Pro Trp Glu Thr Asn Asp 835 840 845 Thr Leu Phe Ser Val Val Ala Leu Pro Trp Leu Ser Glu Gly Glu His 850 855 860 Val Val Asp Val Val Val Glu Asn Ser Ala Ser Arg Ala Asn Leu Ser 865 870 875 880 Leu Arg Val Thr Ala Glu Glu Pro Ile Cys Gly Leu Arg Ala Thr Pro 885 890 895 Ser Pro Glu Ala Arg Val Leu Gln Gly Val Leu Val Arg Tyr Ser Pro 900 905 910 Val Val Glu Ala Gly Ser Asp Met Val Phe Arg Trp Thr Ile Asn Asp 915 920 925 Lys Gln Ser Leu Thr Phe Gln Asn Val Val Phe Asn Val Ile Tyr Gln 930 935 940 Ser Ala Ala Val Phe Lys Leu Ser Leu Thr Ala Ser Asn His Val Ser 945 950 955 960 Asn Val Thr Val Asn Tyr Asn Val Thr Val Glu Arg Met Asn Arg Met 965 970 975 Gln Gly Leu Gln Val Ser Thr Val Pro Ala Val Leu Ser Pro Asn Ala 980 985 990 Thr Leu Val Leu Thr Gly Gly Val Leu Val Asp Ser Ala Val Glu Val 995 1000 1005 Ala Phe Leu Trp Asn Phe Gly Asp Gly Glu Gln Ala Leu His Gln Phe 1010 1015 1020 Gln Pro Pro Tyr Asn Glu Ser Phe Pro Val Pro Asp Pro Ser Val Ala 1025 1030 1035 1040 Gln Val Leu Val Glu His Asn Val Met His Thr Tyr Ala Ala Pro Gly 1045 1050 1055 Glu Tyr Leu Leu Thr Val Leu Ala Ser Asn Ala Phe Glu Asn Leu Thr 1060 1065 1070 Gln Gln Val Pro Val Ser Val Arg Ala Ser Leu Pro Ser Val Ala Val 1075 1080 1085 Gly Val Ser Asp Gly Val Leu Val Ala Gly Arg Pro Val Thr Phe Tyr 1090 1095 1100 Pro His Pro Leu Pro Ser Pro Gly Gly Val Leu Tyr Thr Trp Asp Phe 1105 1110 1115 1120 Gly Asp Gly Ser Pro Val Leu Thr Gln Ser Gln Pro Ala Ala Asn His 1125 1130 1135 Thr Tyr Ala Ser Arg Gly Thr Tyr His Val Arg Leu Glu Val Asn Asn 1140 1145 1150 Thr Val Ser Gly Ala Ala Ala Gln Ala Asp Val Arg Val Phe Glu Glu 1155 1160 1165 Leu Arg Gly Leu Ser Val Asp Met Ser Leu Ala Val Glu Gln Gly Ala 1170 1175 1180 Pro Val Val Val Ser Ala Ala Val Gln Thr Gly Asp Asn Ile Thr Trp 1185 1190 1195 1200 Thr Phe Asp Met Gly Asp Gly Thr Val Leu Ser Gly Pro Glu Ala Thr 1205 1210 1215 Val Glu His Val Tyr Leu Arg Ala Gln Asn Cys Thr Val Thr Val Gly 1220 1225 1230 Ala Ala Ser Pro Ala Gly His Leu Ala Arg Ser Leu His Val Leu Val 1235 1240 1245 Phe Val Leu Glu Val Leu Arg Val Glu Pro Ala Ala Cys Ile Pro Thr 1250 1255 1260 Gln Pro Asp Ala Arg Leu Thr Ala Tyr Val Thr Gly Asn Pro Ala His 1265 1270 1275 1280 Tyr Leu Phe Asp Trp Thr Phe Gly Asp Gly Ser Ser Asn Thr Thr Val 1285 1290 1295 Arg Gly Cys Pro Thr Val Thr His Asn Phe Thr Arg Ser Gly Thr Phe 1300 1305 1310 Pro Leu Ala Leu Val Leu Ser Ser Arg Val Asn Arg Ala His Tyr Phe 1315 1320 1325 Thr Ser Ile Cys Val Glu Pro Glu Val Gly Asn Val Thr Leu Gln Pro 1330 1335 1340 Glu Arg Gln Phe Val Gln Leu Gly Asp Glu Ala Trp Leu Val Ala Cys 1345 1350 1355 1360 Ala Trp Pro Pro Phe Pro Tyr Arg Tyr Thr Trp Asp Phe Gly Thr Glu 1365 1370 1375 Glu Ala Ala Pro Thr Arg Ala Arg Gly Pro Glu Val Thr Phe Ile Tyr 1380 1385 1390 Arg Asp Pro Gly Ser Tyr Leu Val Thr Val Thr Ala Ser Asn Asn Ile 1395 1400 1405 Ser Ala Ala Asn Asp Ser Ala Leu Val Glu Val Gln Glu Pro Val Leu 1410 1415 1420 Val Thr Ser Ile Lys Val Asn Gly Ser Leu Gly Leu Glu Leu Gln Gln 1425 1430 1435 1440 Pro Tyr Leu Phe Ser Ala Val Gly Arg Gly Arg Pro Ala Ser Tyr Leu 1445 1450 1455 Trp Asp Leu Gly Asp Gly Gly Trp Leu Glu Gly Pro Glu Val Thr His 1460 1465 1470 Ala Tyr Asn Ser Thr Gly Asp Phe Thr Val Arg Val Ala Gly Trp Asn 1475 1480 1485 Glu Val Ser Arg Ser Glu Ala Trp Leu Asn Val Thr Val Lys Arg Arg 1490 1495 1500 Val Arg Gly Leu Val Val Asn Ala Ser Arg Thr Val Val Pro Leu Asn 1505 1510 1515 1520 Gly Ser Val Ser Phe Ser Thr Ser Leu Glu Ala Gly Ser Asp Val Arg 1525 1530 1535 Tyr Ser Trp Val Leu Cys Asp Arg Cys Thr Pro Ile Pro Gly Gly Pro 1540 1545 1550 Thr Ile Ser Tyr Thr Phe Arg Ser Val Gly Thr Phe Asn Ile Ile Val 1555 1560 1565 Thr Ala Glu Asn Glu Val Gly Ser Ala Gln Asp Ser Ile Phe Val Tyr 1570 1575 1580 Val Leu Gln Leu Ile Glu Gly Leu Gln Val Val Gly Gly Gly Arg Tyr 1585 1590 1595 1600 Phe Pro Thr Asn His Thr Val Gln Leu Gln Ala Val Val Arg Asp Gly 1605 1610 1615 Thr Asn Val Ser Tyr Ser Trp Thr Ala Trp Arg Asp Arg Gly Pro Ala 1620 1625 1630 Leu Ala Gly Ser Gly Lys Gly Phe Ser Leu Thr Val Leu Glu Ala Gly 1635 1640 1645 Thr Tyr His Val Gln Leu Arg Ala Thr Asn Met Leu Gly Ser Ala Trp 1650 1655 1660 Ala Asp Cys Thr Met Asp Phe Val Glu Pro Val Gly Trp Leu Met Val 1665 1670 1675 1680 Thr Ala Ser Pro Asn Pro Ala Ala Val Asn Thr Ser Val Thr Leu Ser 1685 1690 1695 Ala Glu Leu Ala Gly Gly Ser Gly Val Val Tyr Thr Trp Ser Leu Glu 1700 1705 1710 Glu Gly Leu Ser Trp Glu Thr Ser Glu Pro Phe Thr Thr His Ser Phe 1715 1720 1725 Pro Thr Pro Gly Leu His Leu Val Thr Met Thr Ala Gly Asn Pro Leu 1730 1735 1740 Gly Ser Ala Asn Ala Thr Val Glu Val Asp Val Gln Val Pro Val Ser 1745 1750 1755 1760 Gly Leu Ser Ile Arg Ala Ser Glu Pro Gly Gly Ser Phe Val Ala Ala 1765 1770 1775 Gly Ser Ser Val Pro Phe Trp Gly Gln Leu Ala Thr Gly Thr Asn Val 1780 1785 1790 Ser Trp Cys Trp Ala Val Pro Gly Gly Ser Ser Lys Arg Gly Pro His 1795 1800 1805 Val Thr Met Val Phe Pro Asp Ala Gly Thr Phe Ser Ile Arg Leu Asn 1810 1815 1820 Ala Ser Asn Ala Val Ser Trp Val Ser Ala Thr Tyr Asn Leu Thr Ala 1825 1830 1835 1840 Glu Glu Pro Ile Val Gly Leu Val Leu Trp Ala Ser Ser Lys Val Val 1845 1850 1855 Ala Pro Gly Gln Leu Val His Phe Gln Ile Leu Leu Ala Ala Gly Ser 1860 1865 1870 Ala Val Thr Phe Arg Leu Gln Val Gly Gly Ala Asn Pro Glu Val Leu 1875 1880 1885 Pro Gly Pro Arg Phe Ser His Ser Phe Pro Arg Val Gly Asp His Val 1890 1895 1900 Val Ser Val Arg Gly Lys Asn His Val Ser Trp Ala Gln Ala Gln Val 1905 1910 1915 1920 Arg Ile Val Val Leu Glu Ala Val Ser Gly Leu Gln Met Pro Asn Cys 1925 1930 1935 Cys Glu Pro Gly Ile Ala Thr Gly Thr Glu Arg Asn Phe Thr Ala Arg 1940 1945 1950 Val Gln Arg Gly Ser Arg Val Ala Tyr Ala Trp Tyr Phe Ser Leu Gln 1955 1960 1965 Lys Val Gln Gly Asp Ser Leu Val Ile Leu Ser Gly Arg Asp Val Thr 1970 1975 1980 Tyr Thr Pro Val Ala Ala Gly Leu Leu Glu Ile Gln Val Arg Ala Phe 1985 1990 1995 2000 Asn Ala Leu Gly Ser Glu Asn Arg Thr Leu Val Leu Glu Val Gln Asp 2005 2010 2015 Ala Val Gln Tyr Val Ala Leu Gln Ser Gly Pro Cys Phe Thr Asn Arg 2020 2025 2030 Ser Ala Gln Phe Glu Ala Ala Thr Ser Pro Ser Pro Arg Arg Val Ala 2035 2040 2045 Tyr His Trp Asp Phe Gly Asp Gly Ser Pro Gly Gln Asp Thr Asp Glu 2050 2055 2060 Pro Arg Ala Glu His Ser Tyr Leu Arg Pro Gly Asp Tyr Arg Val Gln 2065 2070 2075 2080 Val Asn Ala Ser Asn Leu Val Ser Phe Phe Val Ala Gln Ala Thr Val 2085 2090 2095 Thr Val Gln Val Leu Ala Cys Arg Glu Pro Glu Val Asp Val Val Leu 2100 2105 2110 Pro Leu Gln Val Leu Met Arg Arg Ser Gln Arg Asn Tyr Leu Glu Ala 2115 2120 2125 His Val Asp Leu Arg Asp Cys Val Thr Tyr Gln Thr Glu Tyr Arg Trp 2130 2135 2140 Glu Val Tyr Arg Thr Ala Ser Cys Gln Arg Pro Gly Arg Pro Ala Arg 2145 2150 2155 2160 Val Ala Leu Pro Gly Val Asp Val Ser Arg Pro Arg Leu Val Leu Pro 2165 2170 2175 Arg Leu Ala Leu Pro Val Gly His Tyr Cys Phe Val Phe Val Val Ser 2180 2185 2190 Phe Gly Asp Thr Pro Leu Thr Gln Ser Ile Gln Ala Asn Val Thr Val 2195 2200 2205 Ala Pro Glu Arg Leu Val Pro Ile Ile Glu Gly Gly Ser Tyr Arg Val 2210 2215 2220 Trp Ser Asp Thr Arg Asp Leu Val Leu Asp Gly Ser Glu Ser Tyr Asp 2225 2230 2235 2240 Pro Asn Leu Glu Asp Gly Asp Gln Thr Pro Leu Ser Phe His Trp Ala 2245 2250 2255 Cys Val Ala Ser Thr Gln Arg Glu Ala Gly Gly Cys Ala Leu Asn Phe 2260 2265 2270 Gly Pro Arg Gly Ser Ser Thr Val Thr Ile Pro Arg Glu Arg Leu Ala 2275 2280 2285 Ala Gly Val Glu Tyr Thr Phe Ser Leu Thr Val Trp Lys Ala Gly Arg 2290 2295 2300 Lys Glu Glu Ala Thr Asn Gln Thr Val Leu Ile Arg Ser Gly Arg Val 2305 2310 2315 2320 Pro Ile Val Ser Leu Glu Cys Val Ser Cys Lys Ala Gln Ala Val Tyr 2325 2330 2335 Glu Val Ser Arg Ser Ser Tyr Val Tyr Leu Glu Gly Arg Cys Leu Asn 2340 2345 2350 Cys Ser Ser Gly Ser Lys Arg Gly Arg Trp Ala Ala Arg Thr Phe Ser 2355 2360 2365 Asn Lys Thr Leu Val Leu Asp Glu Thr Thr Thr Ser Thr Gly Ser Ala 2370 2375 2380 Gly Met Arg Leu Val Leu Arg Arg Gly Val Leu Arg Asp Gly Glu Gly 2385 2390 2395 2400 Tyr Thr Phe Thr Leu Thr Val Leu Gly Arg Ser Gly Glu Glu Glu Gly 2405 2410 2415 Cys Ala Ser Ile Arg Leu Ser Pro Asn Arg Pro Pro Leu Gly Gly Ser 2420 2425 2430 Cys Arg Leu Phe Pro Leu Gly Ala Val His Ala Leu Thr Thr Lys Val 2435 2440 2445 His Phe Glu Cys Thr Gly Trp His Asp Ala Glu Asp Ala Gly Ala Pro 2450 2455 2460 Leu Val Tyr Ala Leu Leu Leu Arg Arg Cys Arg Gln Gly His Cys Glu 2465 2470 2475 2480 Glu Phe Cys Val Tyr Lys Gly Ser Leu Ser Ser Tyr Gly Ala Val Leu 2485 2490 2495 Pro Pro Gly Phe Arg Pro His Phe Glu Val Gly Leu Ala Val Val Val 2500 2505 2510 Gln Asp Gln Leu Gly Ala Ala Val Val Ala Leu Asn Arg Ser Leu Ala 2515 2520 2525 Ile Thr Leu Pro Glu Pro Asn Gly Ser Ala Thr Gly Leu Thr Val Trp 2530 2535 2540 Leu His Gly Leu Thr Ala Ser Val Leu Pro Gly Leu Leu Arg Gln Ala 2545 2550 2555 2560 Asp Pro Gln His Val Ile Glu Tyr Ser Leu Ala Leu Val Thr Val Leu 2565 2570 2575 Asn Glu Tyr Glu Arg Ala Leu Asp Val Ala Ala Glu Pro Lys His Glu 2580 2585 2590 Arg Gln His Arg Ala Gln Ile Arg Lys Asn Ile Thr Glu Thr Leu Val 2595 2600 2605 Ser Leu Arg Val His Thr Val Asp Asp Ile Gln Gln Ile Ala Ala Ala 2610 2615 2620 Leu Ala Gln Cys Met Gly Pro Ser Arg Glu Leu Val Cys Arg Ser Cys 2625 2630 2635 2640 Leu Lys Gln Thr Leu His Lys Leu Glu Ala Met Met Leu Ile Leu Gln 2645 2650 2655 Ala Glu Thr Thr Ala Gly Thr Val Thr Pro Thr Ala Ile Gly Asp Ser 2660 2665 2670 Ile Leu Asn Ile Thr Gly Asp Leu Ile His Leu Ala Ser Ser Asp Val 2675 2680 2685 Arg Ala Pro Gln Pro Ser Glu Leu Gly Ala Glu Ser Pro Ser Arg Met 2690 2695 2700 Val Ala Ser Gln Ala Tyr Asn Leu Thr Ser Ala Leu Met Arg Ile Leu 2705 2710 2715 2720 Met Arg Ser Arg Val Leu Asn Glu Glu Pro Leu Thr Leu Ala Gly Glu 2725 2730 2735 Glu Ile Val Ala Gln Gly Lys Arg Ser Asp Pro Arg Ser Leu Leu Cys 2740 2745 2750 Tyr Gly Gly Ala Pro Gly Pro Gly Cys His Phe Ser Ile Pro Glu Ala 2755 2760 2765 Phe Ser Gly Ala Leu Ala Asn Leu Ser Asp Val Val Gln Leu Ile Phe 2770 2775 2780 Leu Val Asp Ser Asn Pro Phe Pro Phe Gly Tyr Ile Ser Asn Tyr Thr 2785 2790 2795 2800 Val Ser Thr Lys Val Ala Ser Met Ala Phe Gln Thr Gln Ala Gly Ala 2805 2810 2815 Gln Ile Pro Ile Glu Arg Leu Ala Ser Glu Arg Ala Ile Thr Val Lys 2820 2825 2830 Val Pro Asn Asn Ser Asp Trp Ala Ala Arg Gly His Arg Ser Ser Ala 2835 2840 2845 Asn Ser Ala Asn Ser Val Val Val Gln Pro Gln Ala Ser Val Gly Ala 2850 2855 2860 Val Val Thr Leu Asp Ser Ser Asn Pro Ala Ala Gly Leu His Leu Gln 2865 2870 2875 2880 Leu Asn Tyr Thr Leu Leu Asp Gly His Tyr Leu Ser Glu Glu Pro Glu 2885 2890 2895 Pro Tyr Leu Ala Val Tyr Leu His Ser Glu Pro Arg Pro Asn Glu His 2900 2905 2910 Asn Cys Ser Ala Ser Arg Arg Ile Arg Pro Glu Ser Leu Gln Gly Ala 2915 2920 2925 Asp His Arg Pro Tyr Thr Phe Phe Ile Ser Pro Gly Ser Arg Asp Pro 2930 2935 2940 Ala Gly Ser Tyr His Leu Asn Leu Ser Ser His Phe Arg Trp Ser Ala 2945 2950 2955 2960 Leu Gln Val Ser Val Gly Leu Tyr Thr Ser Leu Cys Gln Tyr Phe Ser 2965 2970 2975 Glu Glu Asp Met Val Trp Arg Thr Glu Gly Leu Leu Pro Leu Glu Glu 2980 2985 2990 Thr Ser Pro Arg Gln Ala Val Cys Leu Thr Arg His Leu Thr Ala Phe 2995 3000 3005 Gly Ala Ser Leu Phe Val Pro Pro Ser His Val Arg Phe Val Phe Pro 3010 3015 3020 Glu Pro Thr Ala Asp Val Asn Tyr Ile Val Met Leu Thr Cys Ala Val 3025 3030 3035 3040 Cys Leu Val Thr Tyr Met Val Met Ala Ala Ile Leu His Lys Leu Asp 3045 3050 3055 Gln Leu Asp Ala Ser Arg Gly Arg Ala Ile Pro Phe Cys Gly Gln Arg 3060 3065 3070 Gly Arg Phe Lys Tyr Glu Ile Leu Val Lys Thr Gly Trp Gly Arg Gly 3075 3080 3085 Ser Gly Thr Thr Ala His Val Gly Ile Met Leu Tyr Gly Val Asp Ser 3090 3095 3100 Arg Ser Gly His Arg His Leu Asp Gly Asp Arg Ala Phe His Arg Asn 3105 3110 3115 3120 Ser Leu Asp Ile Phe Arg Ile Ala Thr Pro His Ser Leu Gly Ser Val 3125 3130 3135 Trp Lys Ile Arg Val Trp His Asp Asn Lys Gly Leu Ser Pro Ala Trp 3140 3145 3150 Phe Leu Gln His Val Ile Val Arg Asp Leu Gln Thr Ala Arg Ser Ala 3155 3160 3165 Phe Phe Leu Val Asn Asp Trp Leu Ser Val Glu Thr Glu Ala Asn Gly 3170 3175 3180 Gly Leu Val Glu Lys Glu Val Leu Ala Ala Ser Asp Ala Ala Leu Leu 3185 3190 3195 3200 Arg Phe Arg Arg Leu Leu Val Ala Glu Leu Gln Arg Gly Phe Phe Asp 3205 3210 3215 Lys His Ile Trp Leu Ser Ile Trp Asp Arg Pro Pro Arg Ser Arg Phe 3220 3225 3230 Thr Arg Ile Gln Arg Ala Thr Cys Cys Val Leu Leu Ile Cys Leu Phe 3235 3240 3245 Leu Gly Ala Asn Ala Val Trp Tyr Gly Ala Val Gly Asp Ser Ala Tyr 3250 3255 3260 Ser Thr Gly His Val Ser Arg Leu Ser Pro Leu Ser Val Asp Thr Val 3265 3270 3275 3280 Ala Val Gly Leu Val Ser Ser Val Val Val Tyr Pro Val Tyr Leu Ala 3285 3290 3295 Ile Leu Phe Leu Phe Arg Met Ser Arg Ser Lys Val Ala Gly Ser Pro 3300 3305 3310 Ser Pro Thr Pro Ala Gly Gln Gln Val Leu Asp Ile Asp Ser Cys Leu 3315 3320 3325 Asp Ser Ser Val Leu Asp Ser Ser Phe Leu Thr Phe Ser Gly Leu His 3330 3335 3340 Ala Glu Ala Phe Val Gly Gln Met Lys Ser Asp Leu Phe Leu Asp Asp 3345 3350 3355 3360 Ser Lys Ser Leu Val Cys Trp Pro Ser Gly Glu Gly Thr Leu Ser Trp 3365 3370 3375 Pro Asp Leu Leu Ser Asp Pro Ser Ile Val Gly Ser Asn Leu Arg Gln 3380 3385 3390 Leu Ala Arg Gly Gln Ala Gly His Gly Leu Gly Pro Glu Glu Asp Gly 3395 3400 3405 Phe Ser Leu Ala Ser Pro Tyr Ser Pro Ala Lys Ser Phe Ser Ala Ser 3410 3415 3420 Asp Glu Asp Leu Ile Gln Gln Val Leu Ala Glu Gly Val Ser Ser Pro 3425 3430 3435 3440 Ala Pro Thr Gln Asp Thr His Met Glu Thr Asp Leu Leu Ser Ser Leu 3445 3450 3455 Ser Ser Thr Pro Gly Glu Lys Thr Glu Thr Leu Ala Leu Gln Arg Leu 3460 3465 3470 Gly Glu Leu Gly Pro Pro Ser Pro Gly Leu Asn Trp Glu Gln Pro Gln 3475 3480 3485 Ala Ala Arg Leu Ser Arg Thr Gly Leu Val Glu Gly Leu Arg Lys Arg 3490 3495 3500 Leu Leu Pro Ala Trp Cys Ala Ser Leu Ala His Gly Leu Ser Leu Leu 3505 3510 3515 3520 Leu Val Ala Val Ala Val Ala Val Ser Gly Trp Val Gly Ala Ser Phe 3525 3530 3535 Pro Pro Gly Val Ser Val Ala Trp Leu Leu Ser Ser Ser Ala Ser Phe 3540 3545 3550 Leu Ala Ser Phe Leu Gly Trp Glu Pro Leu Lys Val Leu Leu Glu Ala 3555 3560 3565 Leu Tyr Phe Ser Leu Val Ala Lys Arg Leu His Pro Asp Glu Asp Asp 3570 3575 3580 Thr Leu Val Glu Ser Pro Ala Val Thr Pro Val Ser Ala Arg Val Pro 3585 3590 3595 3600 Arg Val Arg Pro Pro His Gly Phe Ala Leu Phe Leu Ala Lys Glu Glu 3605 3610 3615 Ala Arg Lys Val Lys Arg Leu His Gly Met Leu Arg Ser Leu Leu Val 3620 3625 3630 Tyr Met Leu Phe Leu Leu Val Thr Leu Leu Ala Ser Tyr Gly Asp Ala 3635 3640 3645 Ser Cys His Gly His Ala Tyr Arg Leu Gln Ser Ala Ile Lys Gln Glu 3650 3655 3660 Leu His Ser Arg Ala Phe Leu Ala Ile Thr Arg Ser Glu Glu Leu Trp 3665 3670 3675 3680 Pro Trp Met Ala His Val Leu Leu Pro Tyr Val His Gly Asn Gln Ser 3685 3690 3695 Ser Pro Glu Leu Gly Pro Pro Arg Leu Arg Gln Val Arg Leu Gln Glu 3700 3705 3710 Ala Leu Tyr Pro Asp Pro Pro Gly Pro Arg Val His Thr Cys Ser Ala 3715 3720 3725 Ala Gly Gly Phe Ser Thr Ser Asp Tyr Asp Val Gly Trp Glu Ser Pro 3730 3735 3740 His Asn Gly Ser Gly Thr Trp Ala Tyr Ser Ala Pro Asp Leu Leu Gly 3745 3750 3755 3760 Ala Trp Ser Trp Gly Ser Cys Ala Val Tyr Asp Ser Gly Gly Tyr Val 3765 3770 3775 Gln Glu Leu Gly Leu Ser Leu Glu Glu Ser Arg Asp Arg Leu Arg Phe 3780 3785 3790 Leu Gln Leu His Asn Trp Leu Asp Asn Arg Ser Arg Ala Val Phe Leu 3795 3800 3805 Glu Leu Thr Arg Tyr Ser Pro Ala Val Gly Leu His Ala Ala Val Thr 3810 3815 3820 Leu Arg Leu Glu Phe Pro Ala Ala Gly Arg Ala Leu Ala Ala Leu Ser 3825 3830 3835 3840 Val Arg Pro Phe Ala Leu Arg Arg Leu Ser Ala Gly Leu Ser Leu Pro 3845 3850 3855 Leu Leu Thr Ser Val Cys Leu Leu Leu Phe Ala Val His Phe Ala Val 3860 3865 3870 Ala Glu Ala Arg Thr Trp His Arg Glu Gly Arg Trp Arg Val Leu Arg 3875 3880 3885 Leu Gly Ala Trp Ala Arg Trp Leu Leu Val Ala Leu Thr Ala Ala Thr 3890 3895 3900 Ala Leu Val Arg Leu Ala Gln Leu Gly Ala Ala Asp Arg Gln Trp Thr 3905 3910 3915 3920 Arg Phe Val Arg Gly Arg Pro Arg Arg Phe Thr Ser Phe Asp Gln Val 3925 3930 3935 Ala His Val Ser Ser Ala Ala Arg Gly Leu Ala Ala Ser Leu Leu Phe 3940 3945 3950 Leu Leu Leu Val Lys Ala Ala Gln His Val Arg Phe Val Arg Gln Trp 3955 3960 3965 Ser Val Phe Gly Lys Thr Leu Cys Arg Ala Leu Pro Glu Leu Leu Gly 3970 3975 3980 Val Thr Leu Gly Leu Val Val Leu Gly Val Ala Tyr Ala Gln Leu Ala 3985 3990 3995 4000 Ile Leu Leu Val Ser Ser Cys Val Asp Ser Leu Trp Ser Val Ala Gln 4005 4010 4015 Ala Leu Leu Val Leu Cys Pro Gly Thr Gly Leu Ser Thr Leu Cys Pro 4020 4025 4030 Ala Glu Ser Trp His Leu Ser Pro Leu Leu Cys Val Gly Leu Trp Ala 4035 4040 4045 Leu Arg Leu Trp Gly Ala Leu Arg Leu Gly Ala Val Ile Leu Arg Trp 4050 4055 4060 Arg Tyr His Ala Leu Arg Gly Glu Leu Tyr Arg Pro Ala Trp Glu Pro 4065 4070 4075 4080 Gln Asp Tyr Glu Met Val Glu Leu Phe Leu Arg Arg Leu Arg Leu Trp 4085 4090 4095 Met Gly Leu Ser Lys Val Lys Glu Phe Arg His Lys Val Arg Phe Glu 4100 4105 4110 Gly Met Glu Pro Leu Pro Ser Arg Ser Ser Arg Gly Ser Lys Val Ser 4115 4120 4125 Pro Asp Val Pro Pro Pro Ser Ala Gly Ser Asp Ala Ser His Pro Ser 4130 4135 4140 Thr Ser Ser Ser Gln Leu Asp Gly Leu Ser Val Ser Leu Gly Arg Leu 4145 4150 4155 4160 Gly Thr Arg Cys Glu Pro Glu Pro Ser Arg Leu Gln Ala Val Phe Glu 4165 4170 4175 Ala Leu Leu Thr Gln Phe Asp Arg Leu Asn Gln Ala Thr Glu Asp Val 4180 4185 4190 Tyr Gln Leu Glu Gln Gln Leu His Ser Leu Gln Gly Arg Arg Ser Ser 4195 4200 4205 Arg Ala Pro Ala Gly Ser Ser Arg Gly Pro Ser Pro Gly Leu Arg Pro 4210 4215 4220 Ala Leu Pro Ser Arg Leu Ala Arg Ala Ser Arg Gly Val Asp Leu Ala 4225 4230 4235 4240 Thr Gly Pro Ser Arg Thr Pro Ser Gly Gln Glu Gln Gly Pro Pro Gln 4245 4250 4255 Gln His Leu Val Leu Leu Pro Gly Gly Gly Gly Pro Trp Ser Arg Ser 4260 4265 4270 Gly His Arg Ser Val Leu Leu Ser Ala Ala Val Lys Ala Glu Gly Gln 4275 4280 4285 Ala Glu Trp Leu His Val Gly Ser Pro Glu Ser Arg Gln Gly His Leu 4290 4295 4300 Ser Val Cys Gly Leu Gln His Phe Lys Glu Ala Val Trp Pro Thr Arg 4305 4310 4315 4320 Thr Gln Gly Pro Leu Pro Ser Ser Leu Gly Lys Asp Thr Ala Val Leu 4325 4330 4335 Asp Gly Phe 14148 base pairs nucleic acid unknown unknown cDNA Homo sapiens CDS 212..13117 misc_feature 212..278 /note= “Probable signal sequence” misc_feature 359..4574 /note= “N-linked glycosylation sites at the following positions 359, 476, 557, 572, 770 misc_feature 4574..8144 /note= ”N-linked glycosylation sites at following locations 4559, 4574, 4631, 4763, 4832 misc_feature 8363..11741 /note= “N-linked glycosylation sites at following locations 8471, 8663, 8732, 8843, 8984 misc_feature 7949..8009 /note= ”Predicted transmembrane domain“ misc_feature 8288..8348 /note= ”Predicted transmembrane domain“ misc_feature 9434..9494 /note= ”Predicted transmembrane domain“ misc_feature 10052..10112 /note= ”Predicted transmembrane domain“ misc_feature 10178..10238 /note= ”Predicted transmembrane domain“ misc_feature 10886..10946 /note= ”Predicted transmembrane domain“ misc_feature 10955..11015 /note= ”Predicted transmembrane domain“ misc_feature 11216..11276 /note= ”Predicted transmembrane domain“ misc_feature 11894..11954 /note= ”Predicted transmembrane domain“ misc_feature 12293..12353 /note= ”Predicted transmembrane domain“ misc_feature 12377..12437 /note= ”Predicted transmembrane domain“ misc_feature 212..278 /note= ”Possible hinge sequence“ misc_feature 279 /note= ”Cleavage site“ 7 GCACTGCAGC GCCAGCGTCC GAGCGGGCGG CCGAGCTCCC GGAGCGGCCT GGCCCCGAGC 60 CCCGAGCGGG CGTCGCTCAG CAGCAGGTCG CGGCCGCGCA GCCCCATCCA GCCCCGCGCC 120 CGCCATGCCG TCCGCGGGCC CCGCCTGAGC TGCGGTCTCC GCGCGCGGGC GGGCCTGGGG 180 ACGGCGGGGC CATGCGCGCG CTGCCCTAAC G ATG CCG CCC GCC GCG CCC GCC 232 Met Pro Pro Ala Ala Pro Ala 4340 4345 CGC CTG GCG CTG GCC CTG GGC CTG GGC CTG TGG CTC GGG GCG CTG GCG 280 Arg Leu Ala Leu Ala Leu Gly Leu Gly Leu Trp Leu Gly Ala Leu Ala 4350 4355 4360 GGG GGC CCC GGG CGC GGC TGC GGG CCC TGC GAG CCC CCC TGC CTC TGC 328 Gly Gly Pro Gly Arg Gly Cys Gly Pro Cys Glu Pro Pro Cys Leu Cys 4365 4370 4375 GGC CCA GCG CCC GGC GCC GCC TGC CGC GTC AAC TGC TCG GGC CGC GGG 376 Gly Pro Ala Pro Gly Ala Ala Cys Arg Val Asn Cys Ser Gly Arg Gly 4380 4385 4390 CTG CGG ACG CTC GGT CCC GCG CTG CGC ATC CCC GCG GAC GCC ACA GCG 424 Leu Arg Thr Leu Gly Pro Ala Leu Arg Ile Pro Ala Asp Ala Thr Ala 4395 4400 4405 4410 CTA GAC GTC TCC CAC AAC CTG CTC CGG GCG CTG GAC GTT GGG CTC CTG 472 Leu Asp Val Ser His Asn Leu Leu Arg Ala Leu Asp Val Gly Leu Leu 4415 4420 4425 GCG AAC CTC TCG GCG CTG GCA GAG CTG GAT ATA AGC AAC AAC AAG ATT 520 Ala Asn Leu Ser Ala Leu Ala Glu Leu Asp Ile Ser Asn Asn Lys Ile 4430 4435 4440 TCT ACG TTA GAA GAA GGA ATA TTT GCT AAT TTA TTT AAT TTA AGT GAA 568 Ser Thr Leu Glu Glu Gly Ile Phe Ala Asn Leu Phe Asn Leu Ser Glu 4445 4450 4455 ATA AAC CTG AGT GGG AAC CCG TTT GAG TGT GAC TGT GGC CTG GCG TGG 616 Ile Asn Leu Ser Gly Asn Pro Phe Glu Cys Asp Cys Gly Leu Ala Trp 4460 4465 4470 CTG CCG CGA TGG GCG GAG GAG CAG CAG GTG CGG GTG GTG CAG CCC GAG 664 Leu Pro Arg Trp Ala Glu Glu Gln Gln Val Arg Val Val Gln Pro Glu 4475 4480 4485 4490 GCA GCC ACG TGT GCT GGG CCT GGC TCC CTG GCT GGC CAG CCT CTG CTT 712 Ala Ala Thr Cys Ala Gly Pro Gly Ser Leu Ala Gly Gln Pro Leu Leu 4495 4500 4505 GGC ATC CCC TTG CTG GAC AGT GGC TGT GGT GAG GAG TAT GTC GCC TGC 760 Gly Ile Pro Leu Leu Asp Ser Gly Cys Gly Glu Glu Tyr Val Ala Cys 4510 4515 4520 CTC CCT GAC AAC AGC TCA GGC ACC GTG GCA GCA GTG TCC TTT TCA GCT 808 Leu Pro Asp Asn Ser Ser Gly Thr Val Ala Ala Val Ser Phe Ser Ala 4525 4530 4535 GCC CAC GAA GGC CTG CTT CAG CCA GAG GCC TGC AGC GCC TTC TGC TTC 856 Ala His Glu Gly Leu Leu Gln Pro Glu Ala Cys Ser Ala Phe Cys Phe 4540 4545 4550 TCC ACC GGC CAG GGC CTC GCA GCC CTC TCG GAG CAG GGC TGG TGC CTG 904 Ser Thr Gly Gln Gly Leu Ala Ala Leu Ser Glu Gln Gly Trp Cys Leu 4555 4560 4565 4570 TGT GGG GCG GCC CAG CCC TCC AGT GCC TCC TTT GCC TGC CTG TCC CTC 952 Cys Gly Ala Ala Gln Pro Ser Ser Ala Ser Phe Ala Cys Leu Ser Leu 4575 4580 4585 TGC TCC GGC CCC CCG CCA CCT CCT GCC CCC ACC TGT AGG GGC CCC ACC 1000 Cys Ser Gly Pro Pro Pro Pro Pro Ala Pro Thr Cys Arg Gly Pro Thr 4590 4595 4600 CTC CTC CAG CAC GTC TTC CCT GCC TCC CCA GGG GCC ACC CTG GTG GGG 1048 Leu Leu Gln His Val Phe Pro Ala Ser Pro Gly Ala Thr Leu Val Gly 4605 4610 4615 CCC CAC GGA CCT CTG GCC TCT GGC CAG CTA GCA GCC TTC CAC ATC GCT 1096 Pro His Gly Pro Leu Ala Ser Gly Gln Leu Ala Ala Phe His Ile Ala 4620 4625 4630 GCC CCG CTC CCT GTC ACT GCC ACA CGC TGG GAC TTC GGA GAC GGC TCC 1144 Ala Pro Leu Pro Val Thr Ala Thr Arg Trp Asp Phe Gly Asp Gly Ser 4635 4640 4645 4650 GCC GAG GTG GAT GCC GCT GGG CCG GCT GCC TCG CAT CGC TAT GTG CTG 1192 Ala Glu Val Asp Ala Ala Gly Pro Ala Ala Ser His Arg Tyr Val Leu 4655 4660 4665 CCT GGG CGC TAT CAC GTG ACG GCC GTG CTG GCC CTG GGG GCC GGC TCA 1240 Pro Gly Arg Tyr His Val Thr Ala Val Leu Ala Leu Gly Ala Gly Ser 4670 4675 4680 GCC CTG CTG GGG ACA GAC GTG CAG GTG GAA GCG GCA CCT GCC GCC CTG 1288 Ala Leu Leu Gly Thr Asp Val Gln Val Glu Ala Ala Pro Ala Ala Leu 4685 4690 4695 GAG CTC GTG TGC CCG TCC TCG GTG CAG AGT GAC GAG AGC CTT GAC CTC 1336 Glu Leu Val Cys Pro Ser Ser Val Gln Ser Asp Glu Ser Leu Asp Leu 4700 4705 4710 AGC ATC CAG AAC CGC GGT GGT TCA GGC CTG GAG GCC GCC TAC AGC ATC 1384 Ser Ile Gln Asn Arg Gly Gly Ser Gly Leu Glu Ala Ala Tyr Ser Ile 4715 4720 4725 4730 GTG GCC CTG GGC GAG GAG CCG GCC CGA GCG GTG CAC CCG CTC TGC CCC 1432 Val Ala Leu Gly Glu Glu Pro Ala Arg Ala Val His Pro Leu Cys Pro 4735 4740 4745 TCG GAC ACG GAG ATC TTC CCT GGC AAC GGG CAC TGC TAC CGC CTG GTG 1480 Ser Asp Thr Glu Ile Phe Pro Gly Asn Gly His Cys Tyr Arg Leu Val 4750 4755 4760 GTG GAG AAG GCG GCC TGG CTG CAG GCG CAG GAG CAG TGT CAG GCC TGG 1528 Val Glu Lys Ala Ala Trp Leu Gln Ala Gln Glu Gln Cys Gln Ala Trp 4765 4770 4775 GCC GGG GCC GCC CTG GCA ATG GTG GAC AGT CCC GCC GTG CAG CGC TTC 1576 Ala Gly Ala Ala Leu Ala Met Val Asp Ser Pro Ala Val Gln Arg Phe 4780 4785 4790 CTG GTC TCC CGG GTC ACC AGG AGC CTA GAC GTG TGG ATC GGC TTC TCG 1624 Leu Val Ser Arg Val Thr Arg Ser Leu Asp Val Trp Ile Gly Phe Ser 4795 4800 4805 4810 ACT GTG CAG GGG GTG GAG GTG GGC CCA GCG CCG CAG GGC GAG GCC TTC 1672 Thr Val Gln Gly Val Glu Val Gly Pro Ala Pro Gln Gly Glu Ala Phe 4815 4820 4825 AGC CTG GAG AGC TGC CAG AAC TGG CTG CCC GGG GAG CCA CAC CCA GCC 1720 Ser Leu Glu Ser Cys Gln Asn Trp Leu Pro Gly Glu Pro His Pro Ala 4830 4835 4840 ACA GCC GAG CAC TGC GTC CGG CTC GGG CCC ACC GGG TGG TGT AAC ACC 1768 Thr Ala Glu His Cys Val Arg Leu Gly Pro Thr Gly Trp Cys Asn Thr 4845 4850 4855 GAC CTG TGC TCA GCG CCG CAC AGC TAC GTC TGC GAG CTG CAG CCC GGA 1816 Asp Leu Cys Ser Ala Pro His Ser Tyr Val Cys Glu Leu Gln Pro Gly 4860 4865 4870 GGC CCA GTG CAG GAT GCC GAG AAC CTC CTC GTG GGA GCG CCC AGT GGG 1864 Gly Pro Val Gln Asp Ala Glu Asn Leu Leu Val Gly Ala Pro Ser Gly 4875 4880 4885 4890 GAC CTG CAG GGA CCC CTG ACG CCT CTG GCA CAG CAG GAC GGC CTC TCA 1912 Asp Leu Gln Gly Pro Leu Thr Pro Leu Ala Gln Gln Asp Gly Leu Ser 4895 4900 4905 GCC CCG CAC GAG CCC GTG GAG GTC ATG GTA TTC CCG GGC CTG CGT CTG 1960 Ala Pro His Glu Pro Val Glu Val Met Val Phe Pro Gly Leu Arg Leu 4910 4915 4920 AGC CGT GAA GCC TTC CTC ACC ACG GCC GAA TTT GGG ACC CAG GAG CTC 2008 Ser Arg Glu Ala Phe Leu Thr Thr Ala Glu Phe Gly Thr Gln Glu Leu 4925 4930 4935 CGG CGG CCC GCC CAG CTG CGG CTG CAG GTG TAC CGG CTC CTC AGC ACA 2056 Arg Arg Pro Ala Gln Leu Arg Leu Gln Val Tyr Arg Leu Leu Ser Thr 4940 4945 4950 GCA GGG ACC CCG GAG AAC GGC AGC GAG CCT GAG AGC AGG TCC CCG GAC 2104 Ala Gly Thr Pro Glu Asn Gly Ser Glu Pro Glu Ser Arg Ser Pro Asp 4955 4960 4965 4970 AAC AGG ACC CAG CTG GCC CCC GCG TGC ATG CCA GGG GGA CGC TGG TGC 2152 Asn Arg Thr Gln Leu Ala Pro Ala Cys Met Pro Gly Gly Arg Trp Cys 4975 4980 4985 CCT GGA GCC AAC ATC TGC TTG CCG CTG GAC GCC TCT TGC CAC CCC CAG 2200 Pro Gly Ala Asn Ile Cys Leu Pro Leu Asp Ala Ser Cys His Pro Gln 4990 4995 5000 GCC TGC GCC AAT GGC TGC ACG TCA GGG CCA GGG CTA CCC GGG GCC CCC 2248 Ala Cys Ala Asn Gly Cys Thr Ser Gly Pro Gly Leu Pro Gly Ala Pro 5005 5010 5015 TAT GCG CTA TGG AGA GAG TTC CTC TTC TCC GTT GCC GCG GGG CCC CCC 2296 Tyr Ala Leu Trp Arg Glu Phe Leu Phe Ser Val Ala Ala Gly Pro Pro 5020 5025 5030 GCG CAG TAC TCG GTC ACC CTC CAC GGC CAG GAT GTC CTC ATG CTC CCT 2344 Ala Gln Tyr Ser Val Thr Leu His Gly Gln Asp Val Leu Met Leu Pro 5035 5040 5045 5050 GGT GAC CTC GTT GGC TTG CAG CAC GAC GCT GGC CCT GGC GCC CTC CTG 2392 Gly Asp Leu Val Gly Leu Gln His Asp Ala Gly Pro Gly Ala Leu Leu 5055 5060 5065 CAC TGC TCG CCG GCT CCC GGC CAC CCT GGT CCC CAG GCC CCG TAC CTC 2440 His Cys Ser Pro Ala Pro Gly His Pro Gly Pro Gln Ala Pro Tyr Leu 5070 5075 5080 TCC GCC AAC GCC TCG TCA TGG CTG CCC CAC TTG CCA GCC CAG CTG GAG 2488 Ser Ala Asn Ala Ser Ser Trp Leu Pro His Leu Pro Ala Gln Leu Glu 5085 5090 5095 GGC ACT TGG GCC TGC CCT GCC TGT GCC CTG CGG CTG CTT GCA GCC ACG 2536 Gly Thr Trp Ala Cys Pro Ala Cys Ala Leu Arg Leu Leu Ala Ala Thr 5100 5105 5110 GAA CAG CTC ACC GTG CTG CTG GGC TTG AGG CCC AAC CCT GGA CTG CGG 2584 Glu Gln Leu Thr Val Leu Leu Gly Leu Arg Pro Asn Pro Gly Leu Arg 5115 5120 5125 5130 ATG CCT GGG CGC TAT GAG GTC CGG GCA GAG GTG GGC AAT GGC GTG TCC 2632 Met Pro Gly Arg Tyr Glu Val Arg Ala Glu Val Gly Asn Gly Val Ser 5135 5140 5145 AGG CAC AAC CTC TCC TGC AGC TTT GAC GTG GTC TCC CCA GTG GCT GGG 2680 Arg His Asn Leu Ser Cys Ser Phe Asp Val Val Ser Pro Val Ala Gly 5150 5155 5160 CTG CGG GTC ATC TAC CCT GCC CCC CGC GAC GGC CGC CTC TAC GTG CCC 2728 Leu Arg Val Ile Tyr Pro Ala Pro Arg Asp Gly Arg Leu Tyr Val Pro 5165 5170 5175 ACC AAC GGC TCA GCC TTG GTG CTC CAG GTG GAC TCT GGT GCC AAC GCC 2776 Thr Asn Gly Ser Ala Leu Val Leu Gln Val Asp Ser Gly Ala Asn Ala 5180 5185 5190 ACG GCC ACG GCT CGC TGG CCT GGG GGC AGT GTC AGC GCC CGC TTT GAG 2824 Thr Ala Thr Ala Arg Trp Pro Gly Gly Ser Val Ser Ala Arg Phe Glu 5195 5200 5205 5210 AAT GTC TGC CCT GCC CTG GTG GCC ACC TTC GTG CCC GGC TGC CCC TGG 2872 Asn Val Cys Pro Ala Leu Val Ala Thr Phe Val Pro Gly Cys Pro Trp 5215 5220 5225 GAG ACC AAC GAT ACC CTG TTC TCA GTG GTA GCA CTG CCG TGG CTC AGT 2920 Glu Thr Asn Asp Thr Leu Phe Ser Val Val Ala Leu Pro Trp Leu Ser 5230 5235 5240 GAG GGG GAG CAC GTG GTG GAC GTG GTG GTG GAA AAC AGC GCC AGC CGG 2968 Glu Gly Glu His Val Val Asp Val Val Val Glu Asn Ser Ala Ser Arg 5245 5250 5255 GCC AAC CTC AGC CTG CGG GTG ACG GCG GAG GAG CCC ATC TGT GGC CTC 3016 Ala Asn Leu Ser Leu Arg Val Thr Ala Glu Glu Pro Ile Cys Gly Leu 5260 5265 5270 CGC GCC ACG CCC AGC CCC GAG GCC CGT GTA CTG CAG GGA GTC CTA GTG 3064 Arg Ala Thr Pro Ser Pro Glu Ala Arg Val Leu Gln Gly Val Leu Val 5275 5280 5285 5290 AGG TAC AGC CCC GTG GTG GAG GCC GGC TCG GAC ATG GTC TTC CGG TGG 3112 Arg Tyr Ser Pro Val Val Glu Ala Gly Ser Asp Met Val Phe Arg Trp 5295 5300 5305 ACC ATC AAC GAC AAG CAG TCC CTG ACC TTC CAG AAC GTG GTC TTC AAT 3160 Thr Ile Asn Asp Lys Gln Ser Leu Thr Phe Gln Asn Val Val Phe Asn 5310 5315 5320 GTC ATT TAT CAG AGC GCG GCG GTC TTC AAG CTC TCA CTG ACG GCC TCC 3208 Val Ile Tyr Gln Ser Ala Ala Val Phe Lys Leu Ser Leu Thr Ala Ser 5325 5330 5335 AAC CAC GTG AGC AAC GTC ACC GTG AAC TAC AAC GTA ACC GTG GAG CGG 3256 Asn His Val Ser Asn Val Thr Val Asn Tyr Asn Val Thr Val Glu Arg 5340 5345 5350 ATG AAC AGG ATG CAG GGT CTG CAG GTC TCC ACA GTG CCG GCC GTG CTG 3304 Met Asn Arg Met Gln Gly Leu Gln Val Ser Thr Val Pro Ala Val Leu 5355 5360 5365 5370 TCC CCC AAT GCC ACA CTG GTA CTG ACG GGT GGT GTG CTG GTG GAC TCA 3352 Ser Pro Asn Ala Thr Leu Val Leu Thr Gly Gly Val Leu Val Asp Ser 5375 5380 5385 GCT GTG GAG GTG GCC TTC CTG TGG AAC TTT GGG GAT GGG GAG CAG GCC 3400 Ala Val Glu Val Ala Phe Leu Trp Asn Phe Gly Asp Gly Glu Gln Ala 5390 5395 5400 CTC CAC CAG TTC CAG CCT CCG TAC AAC GAG TCC TTC CCG GTT CCA GAC 3448 Leu His Gln Phe Gln Pro Pro Tyr Asn Glu Ser Phe Pro Val Pro Asp 5405 5410 5415 CCC TCG GTG GCC CAG GTG CTG GTG GAG CAC AAT GTC ATG CAC ACC TAC 3496 Pro Ser Val Ala Gln Val Leu Val Glu His Asn Val Met His Thr Tyr 5420 5425 5430 GCT GCC CCA GGT GAG TAC CTC CTG ACC GTG CTG GCA TCT AAT GCC TTC 3544 Ala Ala Pro Gly Glu Tyr Leu Leu Thr Val Leu Ala Ser Asn Ala Phe 5435 5440 5445 5450 GAG AAC CTG ACG CAG CAG GTG CCT GTG AGC GTG CGC GCC TCC CTG CCC 3592 Glu Asn Leu Thr Gln Gln Val Pro Val Ser Val Arg Ala Ser Leu Pro 5455 5460 5465 TCC GTG GCT GTG GGT GTG AGT GAC GGC GTC CTG GTG GCC GGC CGG CCC 3640 Ser Val Ala Val Gly Val Ser Asp Gly Val Leu Val Ala Gly Arg Pro 5470 5475 5480 GTC ACC TTC TAC CCG CAC CCG CTG CCC TCG CCT GGG GGT GTT CTT TAC 3688 Val Thr Phe Tyr Pro His Pro Leu Pro Ser Pro Gly Gly Val Leu Tyr 5485 5490 5495 ACG TGG GAC TTC GGG GAC GGC TCC CCT GTC CTG ACC CAG AGC CAG CCG 3736 Thr Trp Asp Phe Gly Asp Gly Ser Pro Val Leu Thr Gln Ser Gln Pro 5500 5505 5510 GCT GCC AAC CAC ACC TAT GCC TCG AGG GGC ACC TAC CAC GTG CGC CTG 3784 Ala Ala Asn His Thr Tyr Ala Ser Arg Gly Thr Tyr His Val Arg Leu 5515 5520 5525 5530 GAG GTC AAC AAC ACG GTG AGC GGT GCG GCG GCC CAG GCG GAT GTG CGC 3832 Glu Val Asn Asn Thr Val Ser Gly Ala Ala Ala Gln Ala Asp Val Arg 5535 5540 5545 GTC TTT GAG GAG CTC CGC GGA CTC AGC GTG GAC ATG AGC CTG GCC GTG 3880 Val Phe Glu Glu Leu Arg Gly Leu Ser Val Asp Met Ser Leu Ala Val 5550 5555 5560 GAG CAG GGC GCC CCC GTG GTG GTC AGC GCC GCG GTG CAG ACG GGC GAC 3928 Glu Gln Gly Ala Pro Val Val Val Ser Ala Ala Val Gln Thr Gly Asp 5565 5570 5575 AAC ATC ACG TGG ACC TTC GAC ATG GGG GAC GGC ACC GTG CTG TCG GGC 3976 Asn Ile Thr Trp Thr Phe Asp Met Gly Asp Gly Thr Val Leu Ser Gly 5580 5585 5590 CCG GAG GCA ACA GTG GAG CAT GTG TAC CTG CGG GCA CAG AAC TGC ACA 4024 Pro Glu Ala Thr Val Glu His Val Tyr Leu Arg Ala Gln Asn Cys Thr 5595 5600 5605 5610 GTG ACC GTG GGT GCG GCC AGC CCC GCC GGC CAC CTG GCC CGG AGC CTG 4072 Val Thr Val Gly Ala Ala Ser Pro Ala Gly His Leu Ala Arg Ser Leu 5615 5620 5625 CAC GTG CTG GTC TTC GTC CTG GAG GTG CTG CGC GTT GAA CCC GCC GCC 4120 His Val Leu Val Phe Val Leu Glu Val Leu Arg Val Glu Pro Ala Ala 5630 5635 5640 TGC ATC CCC ACG CAG CCT GAC GCG CGG CTC ACG GCC TAC GTC ACC GGG 4168 Cys Ile Pro Thr Gln Pro Asp Ala Arg Leu Thr Ala Tyr Val Thr Gly 5645 5650 5655 AAC CCG GCC CAC TAC CTC TTC GAC TGG ACC TTC GGG GAT GGC TCC TCC 4216 Asn Pro Ala His Tyr Leu Phe Asp Trp Thr Phe Gly Asp Gly Ser Ser 5660 5665 5670 AAC ACG ACC GTG CGG GGG TGC CCG ACG GTG ACA CAC AAC TTC ACG CGG 4264 Asn Thr Thr Val Arg Gly Cys Pro Thr Val Thr His Asn Phe Thr Arg 5675 5680 5685 5690 AGC GGC ACG TTC CCC CTG GCG CTG GTG CTG TCC AGC CGC GTG AAC AGG 4312 Ser Gly Thr Phe Pro Leu Ala Leu Val Leu Ser Ser Arg Val Asn Arg 5695 5700 5705 GCG CAT TAC TTC ACC AGC ATC TGC GTG GAG CCA GAG GTG GGC AAC GTC 4360 Ala His Tyr Phe Thr Ser Ile Cys Val Glu Pro Glu Val Gly Asn Val 5710 5715 5720 ACC CTG CAG CCA GAG AGG CAG TTT GTG CAG CTC GGG GAC GAG GCC TGG 4408 Thr Leu Gln Pro Glu Arg Gln Phe Val Gln Leu Gly Asp Glu Ala Trp 5725 5730 5735 CTG GTG GCA TGT GCC TGG CCC CCG TTC CCC TAC CGC TAC ACC TGG GAC 4456 Leu Val Ala Cys Ala Trp Pro Pro Phe Pro Tyr Arg Tyr Thr Trp Asp 5740 5745 5750 TTT GGC ACC GAG GAA GCC GCC CCC ACC CGT GCC AGG GGC CCT GAG GTG 4504 Phe Gly Thr Glu Glu Ala Ala Pro Thr Arg Ala Arg Gly Pro Glu Val 5755 5760 5765 5770 ACG TTC ATC TAC CGA GAC CCA GGC TCC TAT CTT GTG ACA GTC ACC GCG 4552 Thr Phe Ile Tyr Arg Asp Pro Gly Ser Tyr Leu Val Thr Val Thr Ala 5775 5780 5785 TCC AAC AAC ATC TCT GCT GCC AAT GAC TCA GCC CTG GTG GAG GTG CAG 4600 Ser Asn Asn Ile Ser Ala Ala Asn Asp Ser Ala Leu Val Glu Val Gln 5790 5795 5800 GAG CCC GTG CTG GTC ACC AGC ATC AAG GTC AAT GGC TCC CTT GGG CTG 4648 Glu Pro Val Leu Val Thr Ser Ile Lys Val Asn Gly Ser Leu Gly Leu 5805 5810 5815 GAG CTG CAG CAG CCG TAC CTG TTC TCT GCT GTG GGC CGT GGG CGC CCC 4696 Glu Leu Gln Gln Pro Tyr Leu Phe Ser Ala Val Gly Arg Gly Arg Pro 5820 5825 5830 GCC AGC TAC CTG TGG GAT CTG GGG GAC GGT GGG TGG CTC GAG GGT CCG 4744 Ala Ser Tyr Leu Trp Asp Leu Gly Asp Gly Gly Trp Leu Glu Gly Pro 5835 5840 5845 5850 GAG GTC ACC CAC GCT TAC AAC AGC ACA GGT GAC TTC ACC GTT AGG GTG 4792 Glu Val Thr His Ala Tyr Asn Ser Thr Gly Asp Phe Thr Val Arg Val 5855 5860 5865 GCC GGC TGG AAT GAG GTG AGC CGC AGC GAG GCC TGG CTC AAT GTG ACG 4840 Ala Gly Trp Asn Glu Val Ser Arg Ser Glu Ala Trp Leu Asn Val Thr 5870 5875 5880 GTG AAG CGG CGC GTG CGG GGG CTC GTC GTC AAT GCA AGC CGC ACG GTG 4888 Val Lys Arg Arg Val Arg Gly Leu Val Val Asn Ala Ser Arg Thr Val 5885 5890 5895 GTG CCC CTG AAT GGG AGC GTG AGC TTC AGC ACG TCG CTG GAG GCC GGC 4936 Val Pro Leu Asn Gly Ser Val Ser Phe Ser Thr Ser Leu Glu Ala Gly 5900 5905 5910 AGT GAT GTG CGC TAT TCC TGG GTG CTC TGT GAC CGC TGC ACG CCC ATC 4984 Ser Asp Val Arg Tyr Ser Trp Val Leu Cys Asp Arg Cys Thr Pro Ile 5915 5920 5925 5930 CCT GGG GGT CCT ACC ATC TCT TAC ACC TTC CGC TCC GTG GGC ACC TTC 5032 Pro Gly Gly Pro Thr Ile Ser Tyr Thr Phe Arg Ser Val Gly Thr Phe 5935 5940 5945 AAT ATC ATC GTC ACG GCT GAG AAC GAG GTG GGC TCC GCC CAG GAC AGC 5080 Asn Ile Ile Val Thr Ala Glu Asn Glu Val Gly Ser Ala Gln Asp Ser 5950 5955 5960 ATC TTC GTC TAT GTC CTG CAG CTC ATA GAG GGG CTG CAG GTG GTG GGC 5128 Ile Phe Val Tyr Val Leu Gln Leu Ile Glu Gly Leu Gln Val Val Gly 5965 5970 5975 GGT GGC CGC TAC TTC CCC ACC AAC CAC ACG GTA CAG CTG CAG GCC GTG 5176 Gly Gly Arg Tyr Phe Pro Thr Asn His Thr Val Gln Leu Gln Ala Val 5980 5985 5990 GTT AGG GAT GGC ACC AAC GTC TCC TAC AGC TGG ACT GCC TGG AGG GAC 5224 Val Arg Asp Gly Thr Asn Val Ser Tyr Ser Trp Thr Ala Trp Arg Asp 5995 6000 6005 6010 AGG GGC CCG GCC CTG GCC GGC AGC GGC AAA GGC TTC TCG CTC ACC GTG 5272 Arg Gly Pro Ala Leu Ala Gly Ser Gly Lys Gly Phe Ser Leu Thr Val 6015 6020 6025 CTC GAG GCC GGC ACC TAC CAT GTG CAG CTG CGG GCC ACC AAC ATG CTG 5320 Leu Glu Ala Gly Thr Tyr His Val Gln Leu Arg Ala Thr Asn Met Leu 6030 6035 6040 GGC AGC GCC TGG GCC GAC TGC ACC ATG GAC TTC GTG GAG CCT GTG GGG 5368 Gly Ser Ala Trp Ala Asp Cys Thr Met Asp Phe Val Glu Pro Val Gly 6045 6050 6055 TGG CTG ATG GTG ACC GCC TCC CCG AAC CCA GCT GCC GTC AAC ACA AGC 5416 Trp Leu Met Val Thr Ala Ser Pro Asn Pro Ala Ala Val Asn Thr Ser 6060 6065 6070 GTC ACC CTC AGT GCC GAG CTG GCT GGT GGC AGT GGT GTC GTA TAC ACT 5464 Val Thr Leu Ser Ala Glu Leu Ala Gly Gly Ser Gly Val Val Tyr Thr 6075 6080 6085 6090 TGG TCC TTG GAG GAG GGG CTG AGC TGG GAG ACC TCC GAG CCA TTT ACC 5512 Trp Ser Leu Glu Glu Gly Leu Ser Trp Glu Thr Ser Glu Pro Phe Thr 6095 6100 6105 ACC CAT AGC TTC CCC ACA CCC GGC CTG CAC TTG GTC ACC ATG ACG GCA 5560 Thr His Ser Phe Pro Thr Pro Gly Leu His Leu Val Thr Met Thr Ala 6110 6115 6120 GGG AAC CCG CTG GGC TCA GCC AAC GCC ACC GTG GAA GTG GAT GTG CAG 5608 Gly Asn Pro Leu Gly Ser Ala Asn Ala Thr Val Glu Val Asp Val Gln 6125 6130 6135 GTG CCT GTG AGT GGC CTC AGC ATC AGG GCC AGC GAG CCC GGA GGC AGC 5656 Val Pro Val Ser Gly Leu Ser Ile Arg Ala Ser Glu Pro Gly Gly Ser 6140 6145 6150 TTC GTG GCG GCC GGG TCC TCT GTG CCC TTT TGG GGG CAG CTG GCC ACG 5704 Phe Val Ala Ala Gly Ser Ser Val Pro Phe Trp Gly Gln Leu Ala Thr 6155 6160 6165 6170 GGC ACC AAT GTG AGC TGG TGC TGG GCT GTG CCC GGC GGC AGC AGC AAG 5752 Gly Thr Asn Val Ser Trp Cys Trp Ala Val Pro Gly Gly Ser Ser Lys 6175 6180 6185 CGT GGC CCT CAT GTC ACC ATG GTC TTC CCG GAT GCT GGC ACC TTC TCC 5800 Arg Gly Pro His Val Thr Met Val Phe Pro Asp Ala Gly Thr Phe Ser 6190 6195 6200 ATC CGG CTC AAT GCC TCC AAC GCA GTC AGC TGG GTC TCA GCC ACG TAC 5848 Ile Arg Leu Asn Ala Ser Asn Ala Val Ser Trp Val Ser Ala Thr Tyr 6205 6210 6215 AAC CTC ACG GCG GAG GAG CCC ATC GTG GGC CTG GTG CTG TGG GCC AGC 5896 Asn Leu Thr Ala Glu Glu Pro Ile Val Gly Leu Val Leu Trp Ala Ser 6220 6225 6230 AGC AAG GTG GTG GCG CCC GGG CAG CTG GTC CAT TTT CAG ATC CTG CTG 5944 Ser Lys Val Val Ala Pro Gly Gln Leu Val His Phe Gln Ile Leu Leu 6235 6240 6245 6250 GCT GCC GGC TCA GCT GTC ACC TTC CGC CTG CAG GTC GGC GGG GCC AAC 5992 Ala Ala Gly Ser Ala Val Thr Phe Arg Leu Gln Val Gly Gly Ala Asn 6255 6260 6265 CCC GAG GTG CTC CCC GGG CCC CGT TTC TCC CAC AGC TTC CCC CGC GTC 6040 Pro Glu Val Leu Pro Gly Pro Arg Phe Ser His Ser Phe Pro Arg Val 6270 6275 6280 GGA GAC CAC GTG GTG AGC GTG CGG GGC AAA AAC CAC GTG AGC TGG GCC 6088 Gly Asp His Val Val Ser Val Arg Gly Lys Asn His Val Ser Trp Ala 6285 6290 6295 CAG GCG CAG GTG CGC ATC GTG GTG CTG GAG GCC GTG AGT GGG CTG CAG 6136 Gln Ala Gln Val Arg Ile Val Val Leu Glu Ala Val Ser Gly Leu Gln 6300 6305 6310 ATG CCC AAC TGC TGC GAG CCT GGC ATC GCC ACG GGC ACT GAG AGG AAC 6184 Met Pro Asn Cys Cys Glu Pro Gly Ile Ala Thr Gly Thr Glu Arg Asn 6315 6320 6325 6330 TTC ACA GCC CGC GTG CAG CGC GGC TCT CGG GTC GCC TAC GCC TGG TAC 6232 Phe Thr Ala Arg Val Gln Arg Gly Ser Arg Val Ala Tyr Ala Trp Tyr 6335 6340 6345 TTC TCG CTG CAG AAG GTC CAG GGC GAC TCG CTG GTC ATC CTG TCG GGC 6280 Phe Ser Leu Gln Lys Val Gln Gly Asp Ser Leu Val Ile Leu Ser Gly 6350 6355 6360 CGC GAC GTC ACC TAC ACG CCC GTG GCC GCG GGG CTG TTG GAG ATC CAG 6328 Arg Asp Val Thr Tyr Thr Pro Val Ala Ala Gly Leu Leu Glu Ile Gln 6365 6370 6375 GTG CGC GCC TTC AAC GCC CTG GGC AGT GAG AAC CGC ACG CTG GTG CTG 6376 Val Arg Ala Phe Asn Ala Leu Gly Ser Glu Asn Arg Thr Leu Val Leu 6380 6385 6390 GAG GTT CAG GAC GCC GTC CAG TAT GTG GCC CTG CAG AGC GGC CCC TGC 6424 Glu Val Gln Asp Ala Val Gln Tyr Val Ala Leu Gln Ser Gly Pro Cys 6395 6400 6405 6410 TTC ACC AAC CGC TCG GCG CAG TTT GAG GCC GCC ACC AGC CCC AGC CCC 6472 Phe Thr Asn Arg Ser Ala Gln Phe Glu Ala Ala Thr Ser Pro Ser Pro 6415 6420 6425 CGG CGT GTG GCC TAC CAC TGG GAC TTT GGG GAT GGG TCG CCA GGG CAG 6520 Arg Arg Val Ala Tyr His Trp Asp Phe Gly Asp Gly Ser Pro Gly Gln 6430 6435 6440 GAC ACA GAT GAG CCC AGG GCC GAG CAC TCC TAC CTG AGG CCT GGG GAC 6568 Asp Thr Asp Glu Pro Arg Ala Glu His Ser Tyr Leu Arg Pro Gly Asp 6445 6450 6455 TAC CGC GTG CAG GTG AAC GCC TCC AAC CTG GTG AGC TTC TTC GTG GCG 6616 Tyr Arg Val Gln Val Asn Ala Ser Asn Leu Val Ser Phe Phe Val Ala 6460 6465 6470 CAG GCC ACG GTG ACC GTC CAG GTG CTG GCC TGC CGG GAG CCG GAG GTG 6664 Gln Ala Thr Val Thr Val Gln Val Leu Ala Cys Arg Glu Pro Glu Val 6475 6480 6485 6490 GAC GTG GTC CTG CCC CTG CAG GTG CTG ATG CGG CGA TCA CAG CGC AAC 6712 Asp Val Val Leu Pro Leu Gln Val Leu Met Arg Arg Ser Gln Arg Asn 6495 6500 6505 TAC TTG GAG GCC CAC GTT GAC CTG CGC GAC TGC GTC ACC TAC CAG ACT 6760 Tyr Leu Glu Ala His Val Asp Leu Arg Asp Cys Val Thr Tyr Gln Thr 6510 6515 6520 GAG TAC CGC TGG GAG GTG TAT CGC ACC GCC AGC TGC CAG CGG CCG GGG 6808 Glu Tyr Arg Trp Glu Val Tyr Arg Thr Ala Ser Cys Gln Arg Pro Gly 6525 6530 6535 CGC CCA GCG CGT GTG GCC CTG CCC GGC GTG GAC GTG AGC CGG CCT CGG 6856 Arg Pro Ala Arg Val Ala Leu Pro Gly Val Asp Val Ser Arg Pro Arg 6540 6545 6550 CTG GTG CTG CCG CGG CTG GCG CTG CCT GTG GGG CAC TAC TGC TTT GTG 6904 Leu Val Leu Pro Arg Leu Ala Leu Pro Val Gly His Tyr Cys Phe Val 6555 6560 6565 6570 TTT GTC GTG TCA TTT GGG GAC ACG CCA CTG ACA CAG AGC ATC CAG GCC 6952 Phe Val Val Ser Phe Gly Asp Thr Pro Leu Thr Gln Ser Ile Gln Ala 6575 6580 6585 AAT GTG ACG GTG GCC CCC GAG CGC CTG GTG CCC ATC ATT GAG GGT GGC 7000 Asn Val Thr Val Ala Pro Glu Arg Leu Val Pro Ile Ile Glu Gly Gly 6590 6595 6600 TCA TAC CGC GTG TGG TCA GAC ACA CGG GAC CTG GTG CTG GAT GGG AGC 7048 Ser Tyr Arg Val Trp Ser Asp Thr Arg Asp Leu Val Leu Asp Gly Ser 6605 6610 6615 GAG TCC TAC GAC CCC AAC CTG GAG GAC GGC GAC CAG ACG CCG CTC AGT 7096 Glu Ser Tyr Asp Pro Asn Leu Glu Asp Gly Asp Gln Thr Pro Leu Ser 6620 6625 6630 TTC CAC TGG GCC TGT GTG GCT TCG ACA CAG AGG GAG GCT GGC GGG TGT 7144 Phe His Trp Ala Cys Val Ala Ser Thr Gln Arg Glu Ala Gly Gly Cys 6635 6640 6645 6650 GCG CTG AAC TTT GGG CCC CGC GGG AGC AGC ACG GTC ACC ATT CCA CGG 7192 Ala Leu Asn Phe Gly Pro Arg Gly Ser Ser Thr Val Thr Ile Pro Arg 6655 6660 6665 GAG CGG CTG GCG GCT GGC GTG GAG TAC ACC TTC AGC CTG ACC GTG TGG 7240 Glu Arg Leu Ala Ala Gly Val Glu Tyr Thr Phe Ser Leu Thr Val Trp 6670 6675 6680 AAG GCC GGC CGC AAG GAG GAG GCC ACC AAC CAG ACG GTG CTG ATC CGG 7288 Lys Ala Gly Arg Lys Glu Glu Ala Thr Asn Gln Thr Val Leu Ile Arg 6685 6690 6695 AGT GGC CGG GTG CCC ATT GTG TCC TTG GAG TGT GTG TCC TGC AAG GCA 7336 Ser Gly Arg Val Pro Ile Val Ser Leu Glu Cys Val Ser Cys Lys Ala 6700 6705 6710 CAG GCC GTG TAC GAA GTG AGC CGC AGC TCC TAC GTG TAC TTG GAG GGC 7384 Gln Ala Val Tyr Glu Val Ser Arg Ser Ser Tyr Val Tyr Leu Glu Gly 6715 6720 6725 6730 CGC TGC CTC AAT TGC AGC AGC GGC TCC AAG CGA GGG CGG TGG GCT GCA 7432 Arg Cys Leu Asn Cys Ser Ser Gly Ser Lys Arg Gly Arg Trp Ala Ala 6735 6740 6745 CGT ACG TTC AGC AAC AAG ACG CTG GTG CTG GAT GAG ACC ACC ACA TCC 7480 Arg Thr Phe Ser Asn Lys Thr Leu Val Leu Asp Glu Thr Thr Thr Ser 6750 6755 6760 ACG GGC AGT GCA GGC ATG CGA CTG GTG CTG CGG CGG GGC GTG CTG CGG 7528 Thr Gly Ser Ala Gly Met Arg Leu Val Leu Arg Arg Gly Val Leu Arg 6765 6770 6775 GAC GGC GAG GGA TAC ACC TTC ACG CTC ACG GTG CTG GGC CGC TCT GGC 7576 Asp Gly Glu Gly Tyr Thr Phe Thr Leu Thr Val Leu Gly Arg Ser Gly 6780 6785 6790 GAG GAG GAG GGC TGC GCC TCC ATC CGC CTG TCC CCC AAC CGC CCG CCG 7624 Glu Glu Glu Gly Cys Ala Ser Ile Arg Leu Ser Pro Asn Arg Pro Pro 6795 6800 6805 6810 CTG GGG GGC TCT TGC CGC CTC TTC CCA CTG GGC GCT GTG CAC GCC CTC 7672 Leu Gly Gly Ser Cys Arg Leu Phe Pro Leu Gly Ala Val His Ala Leu 6815 6820 6825 ACC ACC AAG GTG CAC TTC GAA TGC ACG GGC TGG CAT GAC GCG GAG GAT 7720 Thr Thr Lys Val His Phe Glu Cys Thr Gly Trp His Asp Ala Glu Asp 6830 6835 6840 GCT GGC GCC CCG CTG GTG TAC GCC CTG CTG CTG CGG CGC TGT CGC CAG 7768 Ala Gly Ala Pro Leu Val Tyr Ala Leu Leu Leu Arg Arg Cys Arg Gln 6845 6850 6855 GGC CAC TGC GAG GAG TTC TGT GTC TAC AAG GGC AGC CTC TCC AGC TAC 7816 Gly His Cys Glu Glu Phe Cys Val Tyr Lys Gly Ser Leu Ser Ser Tyr 6860 6865 6870 GGA GCC GTG CTG CCC CCG GGT TTC AGG CCA CAC TTC GAG GTG GGC CTG 7864 Gly Ala Val Leu Pro Pro Gly Phe Arg Pro His Phe Glu Val Gly Leu 6875 6880 6885 6890 GCC GTG GTG GTG CAG GAC CAG CTG GGA GCC GCT GTG GTC GCC CTC AAC 7912 Ala Val Val Val Gln Asp Gln Leu Gly Ala Ala Val Val Ala Leu Asn 6895 6900 6905 AGG TCT TTG GCC ATC ACC CTC CCA GAG CCC AAC GGC AGC GCA ACG GGG 7960 Arg Ser Leu Ala Ile Thr Leu Pro Glu Pro Asn Gly Ser Ala Thr Gly 6910 6915 6920 CTC ACA GTC TGG CTG CAC GGG CTC ACC GCT AGT GTG CTC CCA GGG CTG 8008 Leu Thr Val Trp Leu His Gly Leu Thr Ala Ser Val Leu Pro Gly Leu 6925 6930 6935 CTG CGG CAG GCC GAT CCC CAG CAC GTC ATC GAG TAC TCG TTG GCC CTG 8056 Leu Arg Gln Ala Asp Pro Gln His Val Ile Glu Tyr Ser Leu Ala Leu 6940 6945 6950 GTC ACC GTG CTG AAC GAG TAC GAG CGG GCC CTG GAC GTG GCG GCA GAG 8104 Val Thr Val Leu Asn Glu Tyr Glu Arg Ala Leu Asp Val Ala Ala Glu 6955 6960 6965 6970 CCC AAG CAC GAG CGG CAG CAC CGA GCC CAG ATA CGC AAG AAC ATC ACG 8152 Pro Lys His Glu Arg Gln His Arg Ala Gln Ile Arg Lys Asn Ile Thr 6975 6980 6985 GAG ACT CTG GTG TCC CTG AGG GTC CAC ACT GTG GAT GAC ATC CAG CAG 8200 Glu Thr Leu Val Ser Leu Arg Val His Thr Val Asp Asp Ile Gln Gln 6990 6995 7000 ATC GCT GCT GCG CTG GCC CAG TGC ATG GGG CCC AGC AGG GAG CTC GTA 8248 Ile Ala Ala Ala Leu Ala Gln Cys Met Gly Pro Ser Arg Glu Leu Val 7005 7010 7015 TGC CGC TCG TGC CTG AAG CAG ACG CTG CAC AAG CTG GAG GCC ATG ATG 8296 Cys Arg Ser Cys Leu Lys Gln Thr Leu His Lys Leu Glu Ala Met Met 7020 7025 7030 CTC ATC CTG CAG GCA GAG ACC ACC GCG GGC ACC GTG ACG CCC ACC GCC 8344 Leu Ile Leu Gln Ala Glu Thr Thr Ala Gly Thr Val Thr Pro Thr Ala 7035 7040 7045 7050 ATC GGA GAC AGC ATC CTC AAC ATC ACA GGA GAC CTC ATC CAC CTG GCC 8392 Ile Gly Asp Ser Ile Leu Asn Ile Thr Gly Asp Leu Ile His Leu Ala 7055 7060 7065 AGC TCG GAC GTG CGG GCA CCA CAG CCC TCA GAG CTG GGA GCC GAG TCA 8440 Ser Ser Asp Val Arg Ala Pro Gln Pro Ser Glu Leu Gly Ala Glu Ser 7070 7075 7080 CCA TCT CGG ATG GTG GCG TCC CAG GCC TAC AAC CTG ACC TCT GCC CTC 8488 Pro Ser Arg Met Val Ala Ser Gln Ala Tyr Asn Leu Thr Ser Ala Leu 7085 7090 7095 ATG CGC ATC CTC ATG CGC TCC CGC GTG CTC AAC GAG GAG CCC CTG ACG 8536 Met Arg Ile Leu Met Arg Ser Arg Val Leu Asn Glu Glu Pro Leu Thr 7100 7105 7110 CTG GCG GGC GAG GAG ATC GTG GCC CAG GGC AAG CGC TCG GAC CCG CGG 8584 Leu Ala Gly Glu Glu Ile Val Ala Gln Gly Lys Arg Ser Asp Pro Arg 7115 7120 7125 7130 AGC CTG CTG TGC TAT GGC GGC GCC CCA GGG CCT GGC TGC CAC TTC TCC 8632 Ser Leu Leu Cys Tyr Gly Gly Ala Pro Gly Pro Gly Cys His Phe Ser 7135 7140 7145 ATC CCC GAG GCT TTC AGC GGG GCC CTG GCC AAC CTC AGT GAC GTG GTG 8680 Ile Pro Glu Ala Phe Ser Gly Ala Leu Ala Asn Leu Ser Asp Val Val 7150 7155 7160 CAG CTC ATC TTT CTG GTG GAC TCC AAT CCC TTT CCC TTT GGC TAT ATC 8728 Gln Leu Ile Phe Leu Val Asp Ser Asn Pro Phe Pro Phe Gly Tyr Ile 7165 7170 7175 AGC AAC TAC ACC GTC TCC ACC AAG GTG GCC TCG ATG GCA TTC CAG ACA 8776 Ser Asn Tyr Thr Val Ser Thr Lys Val Ala Ser Met Ala Phe Gln Thr 7180 7185 7190 CAG GCC GGC GCC CAG ATC CCC ATC GAG CGG CTG GCC TCA GAG CGC GCC 8824 Gln Ala Gly Ala Gln Ile Pro Ile Glu Arg Leu Ala Ser Glu Arg Ala 7195 7200 7205 7210 ATC ACC GTG AAG GTG CCC AAC AAC TCG GAC TGG GCT GCC CGG GGC CAC 8872 Ile Thr Val Lys Val Pro Asn Asn Ser Asp Trp Ala Ala Arg Gly His 7215 7220 7225 CGC AGC TCC GCC AAC TCC GCC AAC TCC GTT GTG GTC CAG CCC CAG GCC 8920 Arg Ser Ser Ala Asn Ser Ala Asn Ser Val Val Val Gln Pro Gln Ala 7230 7235 7240 TCC GTC GGT GCT GTG GTC ACC CTG GAC AGC AGC AAC CCT GCG GCC GGG 8968 Ser Val Gly Ala Val Val Thr Leu Asp Ser Ser Asn Pro Ala Ala Gly 7245 7250 7255 CTG CAT CTG CAG CTC AAC TAT ACG CTG CTG GAC GGC CAC TAC CTG TCT 9016 Leu His Leu Gln Leu Asn Tyr Thr Leu Leu Asp Gly His Tyr Leu Ser 7260 7265 7270 GAG GAA CCT GAG CCC TAC CTG GCA GTC TAC CTA CAC TCG GAG CCC CGG 9064 Glu Glu Pro Glu Pro Tyr Leu Ala Val Tyr Leu His Ser Glu Pro Arg 7275 7280 7285 7290 CCC AAT GAG CAC AAC TGC TCG GCT AGC AGG AGG ATC CGC CCA GAG TCA 9112 Pro Asn Glu His Asn Cys Ser Ala Ser Arg Arg Ile Arg Pro Glu Ser 7295 7300 7305 CTC CAG GGT GCT GAC CAC CGG CCC TAC ACC TTC TTC ATT TCC CCG GGG 9160 Leu Gln Gly Ala Asp His Arg Pro Tyr Thr Phe Phe Ile Ser Pro Gly 7310 7315 7320 AGC AGA GAC CCA GCG GGG AGT TAC CAT CTG AAC CTC TCC AGC CAC TTC 9208 Ser Arg Asp Pro Ala Gly Ser Tyr His Leu Asn Leu Ser Ser His Phe 7325 7330 7335 CGC TGG TCG GCG CTG CAG GTG TCC GTG GGC CTG TAC ACG TCC CTG TGC 9256 Arg Trp Ser Ala Leu Gln Val Ser Val Gly Leu Tyr Thr Ser Leu Cys 7340 7345 7350 CAG TAC TTC AGC GAG GAG GAC ATG GTG TGG CGG ACA GAG GGG CTG CTG 9304 Gln Tyr Phe Ser Glu Glu Asp Met Val Trp Arg Thr Glu Gly Leu Leu 7355 7360 7365 7370 CCC CTG GAG GAG ACC TCG CCC CGC CAG GCC GTC TGC CTC ACC CGC CAC 9352 Pro Leu Glu Glu Thr Ser Pro Arg Gln Ala Val Cys Leu Thr Arg His 7375 7380 7385 CTC ACC GCC TTC GGC GCC AGC CTC TTC GTG CCC CCA AGC CAT GTC CGC 9400 Leu Thr Ala Phe Gly Ala Ser Leu Phe Val Pro Pro Ser His Val Arg 7390 7395 7400 TTT GTG TTT CCT GAG CCG ACA GCG GAT GTA AAC TAC ATC GTC ATG CTG 9448 Phe Val Phe Pro Glu Pro Thr Ala Asp Val Asn Tyr Ile Val Met Leu 7405 7410 7415 ACA TGT GCT GTG TGC CTG GTG ACC TAC ATG GTC ATG GCC GCC ATC CTG 9496 Thr Cys Ala Val Cys Leu Val Thr Tyr Met Val Met Ala Ala Ile Leu 7420 7425 7430 CAC AAG CTG GAC CAG TTG GAT GCC AGC CGG GGC CGC GCC ATC CCT TTC 9544 His Lys Leu Asp Gln Leu Asp Ala Ser Arg Gly Arg Ala Ile Pro Phe 7435 7440 7445 7450 TGT GGG CAG CGG GGC CGC TTC AAG TAC GAG ATC CTC GTC AAG ACA GGC 9592 Cys Gly Gln Arg Gly Arg Phe Lys Tyr Glu Ile Leu Val Lys Thr Gly 7455 7460 7465 TGG GGC CGG GGC TCA GGT ACC ACG GCC CAC GTG GGC ATC ATG CTG TAT 9640 Trp Gly Arg Gly Ser Gly Thr Thr Ala His Val Gly Ile Met Leu Tyr 7470 7475 7480 GGG GTG GAC AGC CGG AGC GGC CAC CGG CAC CTG GAC GGC GAC AGA GCC 9688 Gly Val Asp Ser Arg Ser Gly His Arg His Leu Asp Gly Asp Arg Ala 7485 7490 7495 TTC CAC CGC AAC AGC CTG GAC ATC TTC CGG ATC GCC ACC CCG CAC AGC 9736 Phe His Arg Asn Ser Leu Asp Ile Phe Arg Ile Ala Thr Pro His Ser 7500 7505 7510 CTG GGT AGC GTG TGG AAG ATC CGA GTG TGG CAC GAC AAC AAA GGG CTC 9784 Leu Gly Ser Val Trp Lys Ile Arg Val Trp His Asp Asn Lys Gly Leu 7515 7520 7525 7530 AGC CCT GCC TGG TTC CTG CAG CAC GTC ATC GTC AGG GAC CTG CAG ACG 9832 Ser Pro Ala Trp Phe Leu Gln His Val Ile Val Arg Asp Leu Gln Thr 7535 7540 7545 GCA CGC AGC GCC TTC TTC CTG GTC AAT GAC TGG CTT TCG GTG GAG ACG 9880 Ala Arg Ser Ala Phe Phe Leu Val Asn Asp Trp Leu Ser Val Glu Thr 7550 7555 7560 GAG GCC AAC GGG GGC CTG GTG GAG AAG GAG GTG CTG GCC GCG AGC GAC 9928 Glu Ala Asn Gly Gly Leu Val Glu Lys Glu Val Leu Ala Ala Ser Asp 7565 7570 7575 GCA GCC CTT TTG CGC TTC CGG CGC CTG CTG GTG GCT GAG CTG CAG CGT 9976 Ala Ala Leu Leu Arg Phe Arg Arg Leu Leu Val Ala Glu Leu Gln Arg 7580 7585 7590 GGC TTC TTT GAC AAG CAC ATC TGG CTC TCC ATA TGG GAC CGG CCG CCT 10024 Gly Phe Phe Asp Lys His Ile Trp Leu Ser Ile Trp Asp Arg Pro Pro 7595 7600 7605 7610 CGT AGC CGT TTC ACT CGC ATC CAG AGG GCC ACC TGC TGC GTT CTC CTC 10072 Arg Ser Arg Phe Thr Arg Ile Gln Arg Ala Thr Cys Cys Val Leu Leu 7615 7620 7625 ATC TGC CTC TTC CTG GGC GCC AAC GCC GTG TGG TAC GGG GCT GTT GGC 10120 Ile Cys Leu Phe Leu Gly Ala Asn Ala Val Trp Tyr Gly Ala Val Gly 7630 7635 7640 GAC TCT GCC TAC AGC ACG GGG CAT GTG TCC AGG CTG AGC CCG CTG AGC 10168 Asp Ser Ala Tyr Ser Thr Gly His Val Ser Arg Leu Ser Pro Leu Ser 7645 7650 7655 GTC GAC ACA GTC GCT GTT GGC CTG GTG TCC AGC GTG GTT GTC TAT CCC 10216 Val Asp Thr Val Ala Val Gly Leu Val Ser Ser Val Val Val Tyr Pro 7660 7665 7670 GTC TAC CTG GCC ATC CTT TTT CTC TTC CGG ATG TCC CGG AGC AAG GTG 10264 Val Tyr Leu Ala Ile Leu Phe Leu Phe Arg Met Ser Arg Ser Lys Val 7675 7680 7685 7690 GCT GGG AGC CCG AGC CCC ACA CCT GCC GGG CAG CAG GTG CTG GAC ATC 10312 Ala Gly Ser Pro Ser Pro Thr Pro Ala Gly Gln Gln Val Leu Asp Ile 7695 7700 7705 GAC AGC TGC CTG GAC TCG TCC GTG CTG GAC AGC TCC TTC CTC ACG TTC 10360 Asp Ser Cys Leu Asp Ser Ser Val Leu Asp Ser Ser Phe Leu Thr Phe 7710 7715 7720 TCA GGC CTC CAC GCT GAG GCC TTT GTT GGA CAG ATG AAG AGT GAC TTG 10408 Ser Gly Leu His Ala Glu Ala Phe Val Gly Gln Met Lys Ser Asp Leu 7725 7730 7735 TTT CTG GAT GAT TCT AAG AGT CTG GTG TGC TGG CCC TCC GGC GAG GGA 10456 Phe Leu Asp Asp Ser Lys Ser Leu Val Cys Trp Pro Ser Gly Glu Gly 7740 7745 7750 ACG CTC AGT TGG CCG GAC CTG CTC AGT GAC CCG TCC ATT GTG GGT AGC 10504 Thr Leu Ser Trp Pro Asp Leu Leu Ser Asp Pro Ser Ile Val Gly Ser 7755 7760 7765 7770 AAT CTG CGG CAG CTG GCA CGG GGC CAG GCG GGC CAT GGG CTG GGC CCA 10552 Asn Leu Arg Gln Leu Ala Arg Gly Gln Ala Gly His Gly Leu Gly Pro 7775 7780 7785 GAG GAG GAC GGC TTC TCC CTG GCC AGC CCC TAC TCG CCT GCC AAA TCC 10600 Glu Glu Asp Gly Phe Ser Leu Ala Ser Pro Tyr Ser Pro Ala Lys Ser 7790 7795 7800 TTC TCA GCA TCA GAT GAA GAC CTG ATC CAG CAG GTC CTT GCC GAG GGG 10648 Phe Ser Ala Ser Asp Glu Asp Leu Ile Gln Gln Val Leu Ala Glu Gly 7805 7810 7815 GTC AGC AGC CCA GCC CCT ACC CAA GAC ACC CAC ATG GAA ACG GAC CTG 10696 Val Ser Ser Pro Ala Pro Thr Gln Asp Thr His Met Glu Thr Asp Leu 7820 7825 7830 CTC AGC AGC CTG TCC AGC ACT CCT GGG GAG AAG ACA GAG ACG CTG GCG 10744 Leu Ser Ser Leu Ser Ser Thr Pro Gly Glu Lys Thr Glu Thr Leu Ala 7835 7840 7845 7850 CTG CAG AGG CTG GGG GAG CTG GGG CCA CCC AGC CCA GGC CTG AAC TGG 10792 Leu Gln Arg Leu Gly Glu Leu Gly Pro Pro Ser Pro Gly Leu Asn Trp 7855 7860 7865 GAA CAG CCC CAG GCA GCG AGG CTG TCC AGG ACA GGA CTG GTG GAG GGT 10840 Glu Gln Pro Gln Ala Ala Arg Leu Ser Arg Thr Gly Leu Val Glu Gly 7870 7875 7880 CTG CGG AAG CGC CTG CTG CCG GCC TGG TGT GCC TCC CTG GCC CAC GGG 10888 Leu Arg Lys Arg Leu Leu Pro Ala Trp Cys Ala Ser Leu Ala His Gly 7885 7890 7895 CTC AGC CTG CTC CTG GTG GCT GTG GCT GTG GCT GTC TCA GGG TGG GTG 10936 Leu Ser Leu Leu Leu Val Ala Val Ala Val Ala Val Ser Gly Trp Val 7900 7905 7910 GGT GCG AGC TTC CCC CCG GGC GTG AGT GTT GCG TGG CTC CTG TCC AGC 10984 Gly Ala Ser Phe Pro Pro Gly Val Ser Val Ala Trp Leu Leu Ser Ser 7915 7920 7925 7930 AGC GCC AGC TTC CTG GCC TCA TTC CTC GGC TGG GAG CCA CTG AAG GTC 11032 Ser Ala Ser Phe Leu Ala Ser Phe Leu Gly Trp Glu Pro Leu Lys Val 7935 7940 7945 TTG CTG GAA GCC CTG TAC TTC TCA CTG GTG GCC AAG CGG CTG CAC CCG 11080 Leu Leu Glu Ala Leu Tyr Phe Ser Leu Val Ala Lys Arg Leu His Pro 7950 7955 7960 GAT GAA GAT GAC ACC CTG GTA GAG AGC CCG GCT GTG ACG CCT GTG AGC 11128 Asp Glu Asp Asp Thr Leu Val Glu Ser Pro Ala Val Thr Pro Val Ser 7965 7970 7975 GCA CGT GTG CCC CGC GTA CGG CCA CCC CAC GGC TTT GCA CTC TTC CTG 11176 Ala Arg Val Pro Arg Val Arg Pro Pro His Gly Phe Ala Leu Phe Leu 7980 7985 7990 GCC AAG GAA GAA GCC CGC AAG GTC AAG AGG CTA CAT GGC ATG CTG CGG 11224 Ala Lys Glu Glu Ala Arg Lys Val Lys Arg Leu His Gly Met Leu Arg 7995 8000 8005 8010 AGC CTC CTG GTG TAC ATG CTT TTT CTG CTG GTG ACC CTG CTG GCC AGC 11272 Ser Leu Leu Val Tyr Met Leu Phe Leu Leu Val Thr Leu Leu Ala Ser 8015 8020 8025 TAT GGG GAT GCC TCA TGC CAT GGG CAC GCC TAC CGT CTG CAA AGC GCC 11320 Tyr Gly Asp Ala Ser Cys His Gly His Ala Tyr Arg Leu Gln Ser Ala 8030 8035 8040 ATC AAG CAG GAG CTG CAC AGC CGG GCC TTC CTG GCC ATC ACG CGG TCT 11368 Ile Lys Gln Glu Leu His Ser Arg Ala Phe Leu Ala Ile Thr Arg Ser 8045 8050 8055 GAG GAG CTC TGG CCA TGG ATG GCC CAC GTG CTG CTG CCC TAC GTC CAC 11416 Glu Glu Leu Trp Pro Trp Met Ala His Val Leu Leu Pro Tyr Val His 8060 8065 8070 GGG AAC CAG TCC AGC CCA GAG CTG GGG CCC CCA CGG CTG CGG CAG GTG 11464 Gly Asn Gln Ser Ser Pro Glu Leu Gly Pro Pro Arg Leu Arg Gln Val 8075 8080 8085 8090 CGG CTG CAG GAA GCA CTC TAC CCA GAC CCT CCC GGC CCC AGG GTC CAC 11512 Arg Leu Gln Glu Ala Leu Tyr Pro Asp Pro Pro Gly Pro Arg Val His 8095 8100 8105 ACG TGC TCG GCC GCA GGA GGC TTC AGC ACC AGC GAT TAC GAC GTT GGC 11560 Thr Cys Ser Ala Ala Gly Gly Phe Ser Thr Ser Asp Tyr Asp Val Gly 8110 8115 8120 TGG GAG AGT CCT CAC AAT GGC TCG GGG ACG TGG GCC TAT TCA GCG CCG 11608 Trp Glu Ser Pro His Asn Gly Ser Gly Thr Trp Ala Tyr Ser Ala Pro 8125 8130 8135 GAT CTG CTG GGG GCA TGG TCC TGG GGC TCC TGT GCC GTG TAT GAC AGC 11656 Asp Leu Leu Gly Ala Trp Ser Trp Gly Ser Cys Ala Val Tyr Asp Ser 8140 8145 8150 GGG GGC TAC GTG CAG GAG CTG GGC CTG AGC CTG GAG GAG AGC CGC GAC 11704 Gly Gly Tyr Val Gln Glu Leu Gly Leu Ser Leu Glu Glu Ser Arg Asp 8155 8160 8165 8170 CGG CTG CGC TTC CTG CAG CTG CAC AAC TGG CTG GAC AAC AGG AGC CGC 11752 Arg Leu Arg Phe Leu Gln Leu His Asn Trp Leu Asp Asn Arg Ser Arg 8175 8180 8185 GCT GTG TTC CTG GAG CTC ACG CGC TAC AGC CCG GCC GTG GGG CTG CAC 11800 Ala Val Phe Leu Glu Leu Thr Arg Tyr Ser Pro Ala Val Gly Leu His 8190 8195 8200 GCC GCC GTC ACG CTG CGC CTC GAG TTC CCG GCG GCC GGC CGC GCC CTG 11848 Ala Ala Val Thr Leu Arg Leu Glu Phe Pro Ala Ala Gly Arg Ala Leu 8205 8210 8215 GCC GCC CTC AGC GTC CGC CCC TTT GCG CTG CGC CGC CTC AGC GCG GGC 11896 Ala Ala Leu Ser Val Arg Pro Phe Ala Leu Arg Arg Leu Ser Ala Gly 8220 8225 8230 CTC TCG CTG CCT CTG CTC ACC TCG GTG TGC CTG CTG CTG TTC GCC GTG 11944 Leu Ser Leu Pro Leu Leu Thr Ser Val Cys Leu Leu Leu Phe Ala Val 8235 8240 8245 8250 CAC TTC GCC GTG GCC GAG GCC CGT ACT TGG CAC AGG GAA GGG CGC TGG 11992 His Phe Ala Val Ala Glu Ala Arg Thr Trp His Arg Glu Gly Arg Trp 8255 8260 8265 CGC GTG CTG CGG CTC GGA GCC TGG GCG CGG TGG CTG CTG GTG GCG CTG 12040 Arg Val Leu Arg Leu Gly Ala Trp Ala Arg Trp Leu Leu Val Ala Leu 8270 8275 8280 ACG GCG GCC ACG GCA CTG GTA CGC CTC GCC CAG CTG GGT GCC GCT GAC 12088 Thr Ala Ala Thr Ala Leu Val Arg Leu Ala Gln Leu Gly Ala Ala Asp 8285 8290 8295 CGC CAG TGG ACC CGT TTC GTG CGC GGC CGC CCG CGC CGC TTC ACT AGC 12136 Arg Gln Trp Thr Arg Phe Val Arg Gly Arg Pro Arg Arg Phe Thr Ser 8300 8305 8310 TTC GAC CAG GTG GCG CAC GTG AGC TCC GCA GCC CGT GGC CTG GCG GCC 12184 Phe Asp Gln Val Ala His Val Ser Ser Ala Ala Arg Gly Leu Ala Ala 8315 8320 8325 8330 TCG CTG CTC TTC CTG CTT TTG GTC AAG GCT GCC CAG CAC GTA CGC TTC 12232 Ser Leu Leu Phe Leu Leu Leu Val Lys Ala Ala Gln His Val Arg Phe 8335 8340 8345 GTG CGC CAG TGG TCC GTC TTT GGC AAG ACA TTA TGC CGA GCT CTG CCA 12280 Val Arg Gln Trp Ser Val Phe Gly Lys Thr Leu Cys Arg Ala Leu Pro 8350 8355 8360 GAG CTC CTG GGG GTC ACC TTG GGC CTG GTG GTG CTC GGG GTA GCC TAC 12328 Glu Leu Leu Gly Val Thr Leu Gly Leu Val Val Leu Gly Val Ala Tyr 8365 8370 8375 GCC CAG CTG GCC ATC CTG CTC GTG TCT TCC TGT GTG GAC TCC CTC TGG 12376 Ala Gln Leu Ala Ile Leu Leu Val Ser Ser Cys Val Asp Ser Leu Trp 8380 8385 8390 AGC GTG GCC CAG GCC CTG TTG GTG CTG TGC CCT GGG ACT GGG CTC TCT 12424 Ser Val Ala Gln Ala Leu Leu Val Leu Cys Pro Gly Thr Gly Leu Ser 8395 8400 8405 8410 ACC CTG TGT CCT GCC GAG TCC TGG CAC CTG TCA CCC CTG CTG TGT GTG 12472 Thr Leu Cys Pro Ala Glu Ser Trp His Leu Ser Pro Leu Leu Cys Val 8415 8420 8425 GGG CTC TGG GCA CTG CGG CTG TGG GGC GCC CTA CGG CTG GGG GCT GTT 12520 Gly Leu Trp Ala Leu Arg Leu Trp Gly Ala Leu Arg Leu Gly Ala Val 8430 8435 8440 ATT CTC CGC TGG CGC TAC CAC GCC TTG CGT GGA GAG CTG TAC CGG CCG 12568 Ile Leu Arg Trp Arg Tyr His Ala Leu Arg Gly Glu Leu Tyr Arg Pro 8445 8450 8455 GCC TGG GAG CCC CAG GAC TAC GAG ATG GTG GAG TTG TTC CTG CGC AGG 12616 Ala Trp Glu Pro Gln Asp Tyr Glu Met Val Glu Leu Phe Leu Arg Arg 8460 8465 8470 CTG CGC CTC TGG ATG GGC CTC AGC AAG GTC AAG GAG TTC CGC CAC AAA 12664 Leu Arg Leu Trp Met Gly Leu Ser Lys Val Lys Glu Phe Arg His Lys 8475 8480 8485 8490 GTC CGC TTT GAA GGG ATG GAG CCG CTG CCC TCT CGC TCC TCC AGG GGC 12712 Val Arg Phe Glu Gly Met Glu Pro Leu Pro Ser Arg Ser Ser Arg Gly 8495 8500 8505 TCC AAG GTA TCC CCG GAT GTG CCC CCA CCC AGC GCT GGC TCC GAT GCC 12760 Ser Lys Val Ser Pro Asp Val Pro Pro Pro Ser Ala Gly Ser Asp Ala 8510 8515 8520 TCG CAC CCC TCC ACC TCC TCC AGC CAG CTG GAT GGG CTG AGC GTG AGC 12808 Ser His Pro Ser Thr Ser Ser Ser Gln Leu Asp Gly Leu Ser Val Ser 8525 8530 8535 CTG GGC CGG CTG GGG ACA AGG TGT GAG CCT GAG CCC TCC CGC CTC CAA 12856 Leu Gly Arg Leu Gly Thr Arg Cys Glu Pro Glu Pro Ser Arg Leu Gln 8540 8545 8550 GCC GTG TTC GAG GCC CTG CTC ACC CAG TTT GAC CGA CTC AAC CAG GCC 12904 Ala Val Phe Glu Ala Leu Leu Thr Gln Phe Asp Arg Leu Asn Gln Ala 8555 8560 8565 8570 ACA GAG GAC GTC TAC CAG CTG GAG CAG CAG CTG CAC AGC CTG CAA GGC 12952 Thr Glu Asp Val Tyr Gln Leu Glu Gln Gln Leu His Ser Leu Gln Gly 8575 8580 8585 CGC AGG AGC AGC CGG GCG CCC GCC GGA TCT TCC CGT GGC CCA TCC CCG 13000 Arg Arg Ser Ser Arg Ala Pro Ala Gly Ser Ser Arg Gly Pro Ser Pro 8590 8595 8600 GGC CTG CGG CCA GCA CTG CCC AGC CGC CTT GCC CGG GCC AGT CGG GGT 13048 Gly Leu Arg Pro Ala Leu Pro Ser Arg Leu Ala Arg Ala Ser Arg Gly 8605 8610 8615 GTG GAC CTG GCC ACT GGC CCC AGC AGG ACA CCC CTT CGG GCC AAG AAC 13096 Val Asp Leu Ala Thr Gly Pro Ser Arg Thr Pro Leu Arg Ala Lys Asn 8620 8625 8630 AAG GTC CAC CCC AGC AGC ACT TAGTCCTCCT TCCTGGCGGG GGTGGGCCGT 13147 Lys Val His Pro Ser Ser Thr 8635 8640 GGAGTCGGAG TGGACACCGC TCAGTATTAC TTTCTGCCGC TGTCAAGGCC GAGGGCCAGG 13207 CAGAATGGCT GCACGTAGGT TCCCCAGAGA GCAGGCAGGG GCATCTGTCT GTCTGTGGGC 13267 TTCAGCACTT TAAAGAGGCT GTGTGGCCAA CCAGGACCCA GGGTCCCCTC CCCAGCTCCC 13327 TTGGGAAGGA CACAGCAGTA TTGGACGGTT TCTAGCCTCT GAGATGCTAA TTTATTTCCC 13387 CGAGTCCTCA GGTACAGCGG GCTGTGCCCG GCCCCACCCC CTGGGCAGAT GTCCCCCACT 13447 GCTAAGGCTG CTGGCTTCAG GGAGGGTTAG CCTGCACCGC CGCCACCCTG CCCCTAAGTT 13507 ATTACCTCTC CAGTTCCTAC CGTACTCCCT GCACCGTCTC ACTGTGTGTC TCGTGTCAGT 13567 AATTTATATG GTGTTAAAAT GTGTATATTT TTGTATGTCA CTATTTTCAC TAGGGCTGAG 13627 GGGCCTGCGC CCAGAGCTGG CCTCCCCCAA CACCTGCTGC GCTTGGTAGG TGTGGTGGCG 13687 TTATGGCAGC CCGGCTGCTG CTTGGATGCG AGCTTGGCCT TGGGCCGGTG CTGGGGGCAC 13747 AGCTGTCTGC CAGGCACTCT CATCACCCCA GAGGCCTTGT CATCCTCCCT TGCCCCAGGC 13807 CAGGTAGCAA GAGAGCAGCG CCCAGGCCTG CTGGCATCAG GTCTGGGCAA GTAGCAGGAC 13867 TAGGCATGTC AGAGGACCCC AGGGTGGTTA GAGGAAAAGA CTCCTCCTGG GGGCTGGCTC 13927 CCAGGGTGGA GGAAGGTGAC TGTGTGTGTG TGTGTGTGCG CGCGCGACGC GCGAGTGTGC 13987 TGTATGGCCC AGGCAGCCTC AAGGCCCTCG GAGCTGGCTG TGCCTGCTTC TGTGTACCAC 14047 TTCTGTGGGC ATGGCCGCTT CTAGAGCCTC GACACCCCCC CAACCCCCGC ACCAAGCAGA 14107 CAAAGTCAAT AAAAGAGCTG TCTGACTGCA AAAAAAAAAA A 14148 4302 amino acids amino acid linear protein 8 Met Pro Pro Ala Ala Pro Ala Arg Leu Ala Leu Ala Leu Gly Leu Gly 1 5 10 15 Leu Trp Leu Gly Ala Leu Ala Gly Gly Pro Gly Arg Gly Cys Gly Pro 20 25 30 Cys Glu Pro Pro Cys Leu Cys Gly Pro Ala Pro Gly Ala Ala Cys Arg 35 40 45 Val Asn Cys Ser Gly Arg Gly Leu Arg Thr Leu Gly Pro Ala Leu Arg 50 55 60 Ile Pro Ala Asp Ala Thr Ala Leu Asp Val Ser His Asn Leu Leu Arg 65 70 75 80 Ala Leu Asp Val Gly Leu Leu Ala Asn Leu Ser Ala Leu Ala Glu Leu 85 90 95 Asp Ile Ser Asn Asn Lys Ile Ser Thr Leu Glu Glu Gly Ile Phe Ala 100 105 110 Asn Leu Phe Asn Leu Ser Glu Ile Asn Leu Ser Gly Asn Pro Phe Glu 115 120 125 Cys Asp Cys Gly Leu Ala Trp Leu Pro Arg Trp Ala Glu Glu Gln Gln 130 135 140 Val Arg Val Val Gln Pro Glu Ala Ala Thr Cys Ala Gly Pro Gly Ser 145 150 155 160 Leu Ala Gly Gln Pro Leu Leu Gly Ile Pro Leu Leu Asp Ser Gly Cys 165 170 175 Gly Glu Glu Tyr Val Ala Cys Leu Pro Asp Asn Ser Ser Gly Thr Val 180 185 190 Ala Ala Val Ser Phe Ser Ala Ala His Glu Gly Leu Leu Gln Pro Glu 195 200 205 Ala Cys Ser Ala Phe Cys Phe Ser Thr Gly Gln Gly Leu Ala Ala Leu 210 215 220 Ser Glu Gln Gly Trp Cys Leu Cys Gly Ala Ala Gln Pro Ser Ser Ala 225 230 235 240 Ser Phe Ala Cys Leu Ser Leu Cys Ser Gly Pro Pro Pro Pro Pro Ala 245 250 255 Pro Thr Cys Arg Gly Pro Thr Leu Leu Gln His Val Phe Pro Ala Ser 260 265 270 Pro Gly Ala Thr Leu Val Gly Pro His Gly Pro Leu Ala Ser Gly Gln 275 280 285 Leu Ala Ala Phe His Ile Ala Ala Pro Leu Pro Val Thr Ala Thr Arg 290 295 300 Trp Asp Phe Gly Asp Gly Ser Ala Glu Val Asp Ala Ala Gly Pro Ala 305 310 315 320 Ala Ser His Arg Tyr Val Leu Pro Gly Arg Tyr His Val Thr Ala Val 325 330 335 Leu Ala Leu Gly Ala Gly Ser Ala Leu Leu Gly Thr Asp Val Gln Val 340 345 350 Glu Ala Ala Pro Ala Ala Leu Glu Leu Val Cys Pro Ser Ser Val Gln 355 360 365 Ser Asp Glu Ser Leu Asp Leu Ser Ile Gln Asn Arg Gly Gly Ser Gly 370 375 380 Leu Glu Ala Ala Tyr Ser Ile Val Ala Leu Gly Glu Glu Pro Ala Arg 385 390 395 400 Ala Val His Pro Leu Cys Pro Ser Asp Thr Glu Ile Phe Pro Gly Asn 405 410 415 Gly His Cys Tyr Arg Leu Val Val Glu Lys Ala Ala Trp Leu Gln Ala 420 425 430 Gln Glu Gln Cys Gln Ala Trp Ala Gly Ala Ala Leu Ala Met Val Asp 435 440 445 Ser Pro Ala Val Gln Arg Phe Leu Val Ser Arg Val Thr Arg Ser Leu 450 455 460 Asp Val Trp Ile Gly Phe Ser Thr Val Gln Gly Val Glu Val Gly Pro 465 470 475 480 Ala Pro Gln Gly Glu Ala Phe Ser Leu Glu Ser Cys Gln Asn Trp Leu 485 490 495 Pro Gly Glu Pro His Pro Ala Thr Ala Glu His Cys Val Arg Leu Gly 500 505 510 Pro Thr Gly Trp Cys Asn Thr Asp Leu Cys Ser Ala Pro His Ser Tyr 515 520 525 Val Cys Glu Leu Gln Pro Gly Gly Pro Val Gln Asp Ala Glu Asn Leu 530 535 540 Leu Val Gly Ala Pro Ser Gly Asp Leu Gln Gly Pro Leu Thr Pro Leu 545 550 555 560 Ala Gln Gln Asp Gly Leu Ser Ala Pro His Glu Pro Val Glu Val Met 565 570 575 Val Phe Pro Gly Leu Arg Leu Ser Arg Glu Ala Phe Leu Thr Thr Ala 580 585 590 Glu Phe Gly Thr Gln Glu Leu Arg Arg Pro Ala Gln Leu Arg Leu Gln 595 600 605 Val Tyr Arg Leu Leu Ser Thr Ala Gly Thr Pro Glu Asn Gly Ser Glu 610 615 620 Pro Glu Ser Arg Ser Pro Asp Asn Arg Thr Gln Leu Ala Pro Ala Cys 625 630 635 640 Met Pro Gly Gly Arg Trp Cys Pro Gly Ala Asn Ile Cys Leu Pro Leu 645 650 655 Asp Ala Ser Cys His Pro Gln Ala Cys Ala Asn Gly Cys Thr Ser Gly 660 665 670 Pro Gly Leu Pro Gly Ala Pro Tyr Ala Leu Trp Arg Glu Phe Leu Phe 675 680 685 Ser Val Ala Ala Gly Pro Pro Ala Gln Tyr Ser Val Thr Leu His Gly 690 695 700 Gln Asp Val Leu Met Leu Pro Gly Asp Leu Val Gly Leu Gln His Asp 705 710 715 720 Ala Gly Pro Gly Ala Leu Leu His Cys Ser Pro Ala Pro Gly His Pro 725 730 735 Gly Pro Gln Ala Pro Tyr Leu Ser Ala Asn Ala Ser Ser Trp Leu Pro 740 745 750 His Leu Pro Ala Gln Leu Glu Gly Thr Trp Ala Cys Pro Ala Cys Ala 755 760 765 Leu Arg Leu Leu Ala Ala Thr Glu Gln Leu Thr Val Leu Leu Gly Leu 770 775 780 Arg Pro Asn Pro Gly Leu Arg Met Pro Gly Arg Tyr Glu Val Arg Ala 785 790 795 800 Glu Val Gly Asn Gly Val Ser Arg His Asn Leu Ser Cys Ser Phe Asp 805 810 815 Val Val Ser Pro Val Ala Gly Leu Arg Val Ile Tyr Pro Ala Pro Arg 820 825 830 Asp Gly Arg Leu Tyr Val Pro Thr Asn Gly Ser Ala Leu Val Leu Gln 835 840 845 Val Asp Ser Gly Ala Asn Ala Thr Ala Thr Ala Arg Trp Pro Gly Gly 850 855 860 Ser Val Ser Ala Arg Phe Glu Asn Val Cys Pro Ala Leu Val Ala Thr 865 870 875 880 Phe Val Pro Gly Cys Pro Trp Glu Thr Asn Asp Thr Leu Phe Ser Val 885 890 895 Val Ala Leu Pro Trp Leu Ser Glu Gly Glu His Val Val Asp Val Val 900 905 910 Val Glu Asn Ser Ala Ser Arg Ala Asn Leu Ser Leu Arg Val Thr Ala 915 920 925 Glu Glu Pro Ile Cys Gly Leu Arg Ala Thr Pro Ser Pro Glu Ala Arg 930 935 940 Val Leu Gln Gly Val Leu Val Arg Tyr Ser Pro Val Val Glu Ala Gly 945 950 955 960 Ser Asp Met Val Phe Arg Trp Thr Ile Asn Asp Lys Gln Ser Leu Thr 965 970 975 Phe Gln Asn Val Val Phe Asn Val Ile Tyr Gln Ser Ala Ala Val Phe 980 985 990 Lys Leu Ser Leu Thr Ala Ser Asn His Val Ser Asn Val Thr Val Asn 995 1000 1005 Tyr Asn Val Thr Val Glu Arg Met Asn Arg Met Gln Gly Leu Gln Val 1010 1015 1020 Ser Thr Val Pro Ala Val Leu Ser Pro Asn Ala Thr Leu Val Leu Thr 1025 1030 1035 1040 Gly Gly Val Leu Val Asp Ser Ala Val Glu Val Ala Phe Leu Trp Asn 1045 1050 1055 Phe Gly Asp Gly Glu Gln Ala Leu His Gln Phe Gln Pro Pro Tyr Asn 1060 1065 1070 Glu Ser Phe Pro Val Pro Asp Pro Ser Val Ala Gln Val Leu Val Glu 1075 1080 1085 His Asn Val Met His Thr Tyr Ala Ala Pro Gly Glu Tyr Leu Leu Thr 1090 1095 1100 Val Leu Ala Ser Asn Ala Phe Glu Asn Leu Thr Gln Gln Val Pro Val 1105 1110 1115 1120 Ser Val Arg Ala Ser Leu Pro Ser Val Ala Val Gly Val Ser Asp Gly 1125 1130 1135 Val Leu Val Ala Gly Arg Pro Val Thr Phe Tyr Pro His Pro Leu Pro 1140 1145 1150 Ser Pro Gly Gly Val Leu Tyr Thr Trp Asp Phe Gly Asp Gly Ser Pro 1155 1160 1165 Val Leu Thr Gln Ser Gln Pro Ala Ala Asn His Thr Tyr Ala Ser Arg 1170 1175 1180 Gly Thr Tyr His Val Arg Leu Glu Val Asn Asn Thr Val Ser Gly Ala 1185 1190 1195 1200 Ala Ala Gln Ala Asp Val Arg Val Phe Glu Glu Leu Arg Gly Leu Ser 1205 1210 1215 Val Asp Met Ser Leu Ala Val Glu Gln Gly Ala Pro Val Val Val Ser 1220 1225 1230 Ala Ala Val Gln Thr Gly Asp Asn Ile Thr Trp Thr Phe Asp Met Gly 1235 1240 1245 Asp Gly Thr Val Leu Ser Gly Pro Glu Ala Thr Val Glu His Val Tyr 1250 1255 1260 Leu Arg Ala Gln Asn Cys Thr Val Thr Val Gly Ala Ala Ser Pro Ala 1265 1270 1275 1280 Gly His Leu Ala Arg Ser Leu His Val Leu Val Phe Val Leu Glu Val 1285 1290 1295 Leu Arg Val Glu Pro Ala Ala Cys Ile Pro Thr Gln Pro Asp Ala Arg 1300 1305 1310 Leu Thr Ala Tyr Val Thr Gly Asn Pro Ala His Tyr Leu Phe Asp Trp 1315 1320 1325 Thr Phe Gly Asp Gly Ser Ser Asn Thr Thr Val Arg Gly Cys Pro Thr 1330 1335 1340 Val Thr His Asn Phe Thr Arg Ser Gly Thr Phe Pro Leu Ala Leu Val 1345 1350 1355 1360 Leu Ser Ser Arg Val Asn Arg Ala His Tyr Phe Thr Ser Ile Cys Val 1365 1370 1375 Glu Pro Glu Val Gly Asn Val Thr Leu Gln Pro Glu Arg Gln Phe Val 1380 1385 1390 Gln Leu Gly Asp Glu Ala Trp Leu Val Ala Cys Ala Trp Pro Pro Phe 1395 1400 1405 Pro Tyr Arg Tyr Thr Trp Asp Phe Gly Thr Glu Glu Ala Ala Pro Thr 1410 1415 1420 Arg Ala Arg Gly Pro Glu Val Thr Phe Ile Tyr Arg Asp Pro Gly Ser 1425 1430 1435 1440 Tyr Leu Val Thr Val Thr Ala Ser Asn Asn Ile Ser Ala Ala Asn Asp 1445 1450 1455 Ser Ala Leu Val Glu Val Gln Glu Pro Val Leu Val Thr Ser Ile Lys 1460 1465 1470 Val Asn Gly Ser Leu Gly Leu Glu Leu Gln Gln Pro Tyr Leu Phe Ser 1475 1480 1485 Ala Val Gly Arg Gly Arg Pro Ala Ser Tyr Leu Trp Asp Leu Gly Asp 1490 1495 1500 Gly Gly Trp Leu Glu Gly Pro Glu Val Thr His Ala Tyr Asn Ser Thr 1505 1510 1515 1520 Gly Asp Phe Thr Val Arg Val Ala Gly Trp Asn Glu Val Ser Arg Ser 1525 1530 1535 Glu Ala Trp Leu Asn Val Thr Val Lys Arg Arg Val Arg Gly Leu Val 1540 1545 1550 Val Asn Ala Ser Arg Thr Val Val Pro Leu Asn Gly Ser Val Ser Phe 1555 1560 1565 Ser Thr Ser Leu Glu Ala Gly Ser Asp Val Arg Tyr Ser Trp Val Leu 1570 1575 1580 Cys Asp Arg Cys Thr Pro Ile Pro Gly Gly Pro Thr Ile Ser Tyr Thr 1585 1590 1595 1600 Phe Arg Ser Val Gly Thr Phe Asn Ile Ile Val Thr Ala Glu Asn Glu 1605 1610 1615 Val Gly Ser Ala Gln Asp Ser Ile Phe Val Tyr Val Leu Gln Leu Ile 1620 1625 1630 Glu Gly Leu Gln Val Val Gly Gly Gly Arg Tyr Phe Pro Thr Asn His 1635 1640 1645 Thr Val Gln Leu Gln Ala Val Val Arg Asp Gly Thr Asn Val Ser Tyr 1650 1655 1660 Ser Trp Thr Ala Trp Arg Asp Arg Gly Pro Ala Leu Ala Gly Ser Gly 1665 1670 1675 1680 Lys Gly Phe Ser Leu Thr Val Leu Glu Ala Gly Thr Tyr His Val Gln 1685 1690 1695 Leu Arg Ala Thr Asn Met Leu Gly Ser Ala Trp Ala Asp Cys Thr Met 1700 1705 1710 Asp Phe Val Glu Pro Val Gly Trp Leu Met Val Thr Ala Ser Pro Asn 1715 1720 1725 Pro Ala Ala Val Asn Thr Ser Val Thr Leu Ser Ala Glu Leu Ala Gly 1730 1735 1740 Gly Ser Gly Val Val Tyr Thr Trp Ser Leu Glu Glu Gly Leu Ser Trp 1745 1750 1755 1760 Glu Thr Ser Glu Pro Phe Thr Thr His Ser Phe Pro Thr Pro Gly Leu 1765 1770 1775 His Leu Val Thr Met Thr Ala Gly Asn Pro Leu Gly Ser Ala Asn Ala 1780 1785 1790 Thr Val Glu Val Asp Val Gln Val Pro Val Ser Gly Leu Ser Ile Arg 1795 1800 1805 Ala Ser Glu Pro Gly Gly Ser Phe Val Ala Ala Gly Ser Ser Val Pro 1810 1815 1820 Phe Trp Gly Gln Leu Ala Thr Gly Thr Asn Val Ser Trp Cys Trp Ala 1825 1830 1835 1840 Val Pro Gly Gly Ser Ser Lys Arg Gly Pro His Val Thr Met Val Phe 1845 1850 1855 Pro Asp Ala Gly Thr Phe Ser Ile Arg Leu Asn Ala Ser Asn Ala Val 1860 1865 1870 Ser Trp Val Ser Ala Thr Tyr Asn Leu Thr Ala Glu Glu Pro Ile Val 1875 1880 1885 Gly Leu Val Leu Trp Ala Ser Ser Lys Val Val Ala Pro Gly Gln Leu 1890 1895 1900 Val His Phe Gln Ile Leu Leu Ala Ala Gly Ser Ala Val Thr Phe Arg 1905 1910 1915 1920 Leu Gln Val Gly Gly Ala Asn Pro Glu Val Leu Pro Gly Pro Arg Phe 1925 1930 1935 Ser His Ser Phe Pro Arg Val Gly Asp His Val Val Ser Val Arg Gly 1940 1945 1950 Lys Asn His Val Ser Trp Ala Gln Ala Gln Val Arg Ile Val Val Leu 1955 1960 1965 Glu Ala Val Ser Gly Leu Gln Met Pro Asn Cys Cys Glu Pro Gly Ile 1970 1975 1980 Ala Thr Gly Thr Glu Arg Asn Phe Thr Ala Arg Val Gln Arg Gly Ser 1985 1990 1995 2000 Arg Val Ala Tyr Ala Trp Tyr Phe Ser Leu Gln Lys Val Gln Gly Asp 2005 2010 2015 Ser Leu Val Ile Leu Ser Gly Arg Asp Val Thr Tyr Thr Pro Val Ala 2020 2025 2030 Ala Gly Leu Leu Glu Ile Gln Val Arg Ala Phe Asn Ala Leu Gly Ser 2035 2040 2045 Glu Asn Arg Thr Leu Val Leu Glu Val Gln Asp Ala Val Gln Tyr Val 2050 2055 2060 Ala Leu Gln Ser Gly Pro Cys Phe Thr Asn Arg Ser Ala Gln Phe Glu 2065 2070 2075 2080 Ala Ala Thr Ser Pro Ser Pro Arg Arg Val Ala Tyr His Trp Asp Phe 2085 2090 2095 Gly Asp Gly Ser Pro Gly Gln Asp Thr Asp Glu Pro Arg Ala Glu His 2100 2105 2110 Ser Tyr Leu Arg Pro Gly Asp Tyr Arg Val Gln Val Asn Ala Ser Asn 2115 2120 2125 Leu Val Ser Phe Phe Val Ala Gln Ala Thr Val Thr Val Gln Val Leu 2130 2135 2140 Ala Cys Arg Glu Pro Glu Val Asp Val Val Leu Pro Leu Gln Val Leu 2145 2150 2155 2160 Met Arg Arg Ser Gln Arg Asn Tyr Leu Glu Ala His Val Asp Leu Arg 2165 2170 2175 Asp Cys Val Thr Tyr Gln Thr Glu Tyr Arg Trp Glu Val Tyr Arg Thr 2180 2185 2190 Ala Ser Cys Gln Arg Pro Gly Arg Pro Ala Arg Val Ala Leu Pro Gly 2195 2200 2205 Val Asp Val Ser Arg Pro Arg Leu Val Leu Pro Arg Leu Ala Leu Pro 2210 2215 2220 Val Gly His Tyr Cys Phe Val Phe Val Val Ser Phe Gly Asp Thr Pro 2225 2230 2235 2240 Leu Thr Gln Ser Ile Gln Ala Asn Val Thr Val Ala Pro Glu Arg Leu 2245 2250 2255 Val Pro Ile Ile Glu Gly Gly Ser Tyr Arg Val Trp Ser Asp Thr Arg 2260 2265 2270 Asp Leu Val Leu Asp Gly Ser Glu Ser Tyr Asp Pro Asn Leu Glu Asp 2275 2280 2285 Gly Asp Gln Thr Pro Leu Ser Phe His Trp Ala Cys Val Ala Ser Thr 2290 2295 2300 Gln Arg Glu Ala Gly Gly Cys Ala Leu Asn Phe Gly Pro Arg Gly Ser 2305 2310 2315 2320 Ser Thr Val Thr Ile Pro Arg Glu Arg Leu Ala Ala Gly Val Glu Tyr 2325 2330 2335 Thr Phe Ser Leu Thr Val Trp Lys Ala Gly Arg Lys Glu Glu Ala Thr 2340 2345 2350 Asn Gln Thr Val Leu Ile Arg Ser Gly Arg Val Pro Ile Val Ser Leu 2355 2360 2365 Glu Cys Val Ser Cys Lys Ala Gln Ala Val Tyr Glu Val Ser Arg Ser 2370 2375 2380 Ser Tyr Val Tyr Leu Glu Gly Arg Cys Leu Asn Cys Ser Ser Gly Ser 2385 2390 2395 2400 Lys Arg Gly Arg Trp Ala Ala Arg Thr Phe Ser Asn Lys Thr Leu Val 2405 2410 2415 Leu Asp Glu Thr Thr Thr Ser Thr Gly Ser Ala Gly Met Arg Leu Val 2420 2425 2430 Leu Arg Arg Gly Val Leu Arg Asp Gly Glu Gly Tyr Thr Phe Thr Leu 2435 2440 2445 Thr Val Leu Gly Arg Ser Gly Glu Glu Glu Gly Cys Ala Ser Ile Arg 2450 2455 2460 Leu Ser Pro Asn Arg Pro Pro Leu Gly Gly Ser Cys Arg Leu Phe Pro 2465 2470 2475 2480 Leu Gly Ala Val His Ala Leu Thr Thr Lys Val His Phe Glu Cys Thr 2485 2490 2495 Gly Trp His Asp Ala Glu Asp Ala Gly Ala Pro Leu Val Tyr Ala Leu 2500 2505 2510 Leu Leu Arg Arg Cys Arg Gln Gly His Cys Glu Glu Phe Cys Val Tyr 2515 2520 2525 Lys Gly Ser Leu Ser Ser Tyr Gly Ala Val Leu Pro Pro Gly Phe Arg 2530 2535 2540 Pro His Phe Glu Val Gly Leu Ala Val Val Val Gln Asp Gln Leu Gly 2545 2550 2555 2560 Ala Ala Val Val Ala Leu Asn Arg Ser Leu Ala Ile Thr Leu Pro Glu 2565 2570 2575 Pro Asn Gly Ser Ala Thr Gly Leu Thr Val Trp Leu His Gly Leu Thr 2580 2585 2590 Ala Ser Val Leu Pro Gly Leu Leu Arg Gln Ala Asp Pro Gln His Val 2595 2600 2605 Ile Glu Tyr Ser Leu Ala Leu Val Thr Val Leu Asn Glu Tyr Glu Arg 2610 2615 2620 Ala Leu Asp Val Ala Ala Glu Pro Lys His Glu Arg Gln His Arg Ala 2625 2630 2635 2640 Gln Ile Arg Lys Asn Ile Thr Glu Thr Leu Val Ser Leu Arg Val His 2645 2650 2655 Thr Val Asp Asp Ile Gln Gln Ile Ala Ala Ala Leu Ala Gln Cys Met 2660 2665 2670 Gly Pro Ser Arg Glu Leu Val Cys Arg Ser Cys Leu Lys Gln Thr Leu 2675 2680 2685 His Lys Leu Glu Ala Met Met Leu Ile Leu Gln Ala Glu Thr Thr Ala 2690 2695 2700 Gly Thr Val Thr Pro Thr Ala Ile Gly Asp Ser Ile Leu Asn Ile Thr 2705 2710 2715 2720 Gly Asp Leu Ile His Leu Ala Ser Ser Asp Val Arg Ala Pro Gln Pro 2725 2730 2735 Ser Glu Leu Gly Ala Glu Ser Pro Ser Arg Met Val Ala Ser Gln Ala 2740 2745 2750 Tyr Asn Leu Thr Ser Ala Leu Met Arg Ile Leu Met Arg Ser Arg Val 2755 2760 2765 Leu Asn Glu Glu Pro Leu Thr Leu Ala Gly Glu Glu Ile Val Ala Gln 2770 2775 2780 Gly Lys Arg Ser Asp Pro Arg Ser Leu Leu Cys Tyr Gly Gly Ala Pro 2785 2790 2795 2800 Gly Pro Gly Cys His Phe Ser Ile Pro Glu Ala Phe Ser Gly Ala Leu 2805 2810 2815 Ala Asn Leu Ser Asp Val Val Gln Leu Ile Phe Leu Val Asp Ser Asn 2820 2825 2830 Pro Phe Pro Phe Gly Tyr Ile Ser Asn Tyr Thr Val Ser Thr Lys Val 2835 2840 2845 Ala Ser Met Ala Phe Gln Thr Gln Ala Gly Ala Gln Ile Pro Ile Glu 2850 2855 2860 Arg Leu Ala Ser Glu Arg Ala Ile Thr Val Lys Val Pro Asn Asn Ser 2865 2870 2875 2880 Asp Trp Ala Ala Arg Gly His Arg Ser Ser Ala Asn Ser Ala Asn Ser 2885 2890 2895 Val Val Val Gln Pro Gln Ala Ser Val Gly Ala Val Val Thr Leu Asp 2900 2905 2910 Ser Ser Asn Pro Ala Ala Gly Leu His Leu Gln Leu Asn Tyr Thr Leu 2915 2920 2925 Leu Asp Gly His Tyr Leu Ser Glu Glu Pro Glu Pro Tyr Leu Ala Val 2930 2935 2940 Tyr Leu His Ser Glu Pro Arg Pro Asn Glu His Asn Cys Ser Ala Ser 2945 2950 2955 2960 Arg Arg Ile Arg Pro Glu Ser Leu Gln Gly Ala Asp His Arg Pro Tyr 2965 2970 2975 Thr Phe Phe Ile Ser Pro Gly Ser Arg Asp Pro Ala Gly Ser Tyr His 2980 2985 2990 Leu Asn Leu Ser Ser His Phe Arg Trp Ser Ala Leu Gln Val Ser Val 2995 3000 3005 Gly Leu Tyr Thr Ser Leu Cys Gln Tyr Phe Ser Glu Glu Asp Met Val 3010 3015 3020 Trp Arg Thr Glu Gly Leu Leu Pro Leu Glu Glu Thr Ser Pro Arg Gln 3025 3030 3035 3040 Ala Val Cys Leu Thr Arg His Leu Thr Ala Phe Gly Ala Ser Leu Phe 3045 3050 3055 Val Pro Pro Ser His Val Arg Phe Val Phe Pro Glu Pro Thr Ala Asp 3060 3065 3070 Val Asn Tyr Ile Val Met Leu Thr Cys Ala Val Cys Leu Val Thr Tyr 3075 3080 3085 Met Val Met Ala Ala Ile Leu His Lys Leu Asp Gln Leu Asp Ala Ser 3090 3095 3100 Arg Gly Arg Ala Ile Pro Phe Cys Gly Gln Arg Gly Arg Phe Lys Tyr 3105 3110 3115 3120 Glu Ile Leu Val Lys Thr Gly Trp Gly Arg Gly Ser Gly Thr Thr Ala 3125 3130 3135 His Val Gly Ile Met Leu Tyr Gly Val Asp Ser Arg Ser Gly His Arg 3140 3145 3150 His Leu Asp Gly Asp Arg Ala Phe His Arg Asn Ser Leu Asp Ile Phe 3155 3160 3165 Arg Ile Ala Thr Pro His Ser Leu Gly Ser Val Trp Lys Ile Arg Val 3170 3175 3180 Trp His Asp Asn Lys Gly Leu Ser Pro Ala Trp Phe Leu Gln His Val 3185 3190 3195 3200 Ile Val Arg Asp Leu Gln Thr Ala Arg Ser Ala Phe Phe Leu Val Asn 3205 3210 3215 Asp Trp Leu Ser Val Glu Thr Glu Ala Asn Gly Gly Leu Val Glu Lys 3220 3225 3230 Glu Val Leu Ala Ala Ser Asp Ala Ala Leu Leu Arg Phe Arg Arg Leu 3235 3240 3245 Leu Val Ala Glu Leu Gln Arg Gly Phe Phe Asp Lys His Ile Trp Leu 3250 3255 3260 Ser Ile Trp Asp Arg Pro Pro Arg Ser Arg Phe Thr Arg Ile Gln Arg 3265 3270 3275 3280 Ala Thr Cys Cys Val Leu Leu Ile Cys Leu Phe Leu Gly Ala Asn Ala 3285 3290 3295 Val Trp Tyr Gly Ala Val Gly Asp Ser Ala Tyr Ser Thr Gly His Val 3300 3305 3310 Ser Arg Leu Ser Pro Leu Ser Val Asp Thr Val Ala Val Gly Leu Val 3315 3320 3325 Ser Ser Val Val Val Tyr Pro Val Tyr Leu Ala Ile Leu Phe Leu Phe 3330 3335 3340 Arg Met Ser Arg Ser Lys Val Ala Gly Ser Pro Ser Pro Thr Pro Ala 3345 3350 3355 3360 Gly Gln Gln Val Leu Asp Ile Asp Ser Cys Leu Asp Ser Ser Val Leu 3365 3370 3375 Asp Ser Ser Phe Leu Thr Phe Ser Gly Leu His Ala Glu Ala Phe Val 3380 3385 3390 Gly Gln Met Lys Ser Asp Leu Phe Leu Asp Asp Ser Lys Ser Leu Val 3395 3400 3405 Cys Trp Pro Ser Gly Glu Gly Thr Leu Ser Trp Pro Asp Leu Leu Ser 3410 3415 3420 Asp Pro Ser Ile Val Gly Ser Asn Leu Arg Gln Leu Ala Arg Gly Gln 3425 3430 3435 3440 Ala Gly His Gly Leu Gly Pro Glu Glu Asp Gly Phe Ser Leu Ala Ser 3445 3450 3455 Pro Tyr Ser Pro Ala Lys Ser Phe Ser Ala Ser Asp Glu Asp Leu Ile 3460 3465 3470 Gln Gln Val Leu Ala Glu Gly Val Ser Ser Pro Ala Pro Thr Gln Asp 3475 3480 3485 Thr His Met Glu Thr Asp Leu Leu Ser Ser Leu Ser Ser Thr Pro Gly 3490 3495 3500 Glu Lys Thr Glu Thr Leu Ala Leu Gln Arg Leu Gly Glu Leu Gly Pro 3505 3510 3515 3520 Pro Ser Pro Gly Leu Asn Trp Glu Gln Pro Gln Ala Ala Arg Leu Ser 3525 3530 3535 Arg Thr Gly Leu Val Glu Gly Leu Arg Lys Arg Leu Leu Pro Ala Trp 3540 3545 3550 Cys Ala Ser Leu Ala His Gly Leu Ser Leu Leu Leu Val Ala Val Ala 3555 3560 3565 Val Ala Val Ser Gly Trp Val Gly Ala Ser Phe Pro Pro Gly Val Ser 3570 3575 3580 Val Ala Trp Leu Leu Ser Ser Ser Ala Ser Phe Leu Ala Ser Phe Leu 3585 3590 3595 3600 Gly Trp Glu Pro Leu Lys Val Leu Leu Glu Ala Leu Tyr Phe Ser Leu 3605 3610 3615 Val Ala Lys Arg Leu His Pro Asp Glu Asp Asp Thr Leu Val Glu Ser 3620 3625 3630 Pro Ala Val Thr Pro Val Ser Ala Arg Val Pro Arg Val Arg Pro Pro 3635 3640 3645 His Gly Phe Ala Leu Phe Leu Ala Lys Glu Glu Ala Arg Lys Val Lys 3650 3655 3660 Arg Leu His Gly Met Leu Arg Ser Leu Leu Val Tyr Met Leu Phe Leu 3665 3670 3675 3680 Leu Val Thr Leu Leu Ala Ser Tyr Gly Asp Ala Ser Cys His Gly His 3685 3690 3695 Ala Tyr Arg Leu Gln Ser Ala Ile Lys Gln Glu Leu His Ser Arg Ala 3700 3705 3710 Phe Leu Ala Ile Thr Arg Ser Glu Glu Leu Trp Pro Trp Met Ala His 3715 3720 3725 Val Leu Leu Pro Tyr Val His Gly Asn Gln Ser Ser Pro Glu Leu Gly 3730 3735 3740 Pro Pro Arg Leu Arg Gln Val Arg Leu Gln Glu Ala Leu Tyr Pro Asp 3745 3750 3755 3760 Pro Pro Gly Pro Arg Val His Thr Cys Ser Ala Ala Gly Gly Phe Ser 3765 3770 3775 Thr Ser Asp Tyr Asp Val Gly Trp Glu Ser Pro His Asn Gly Ser Gly 3780 3785 3790 Thr Trp Ala Tyr Ser Ala Pro Asp Leu Leu Gly Ala Trp Ser Trp Gly 3795 3800 3805 Ser Cys Ala Val Tyr Asp Ser Gly Gly Tyr Val Gln Glu Leu Gly Leu 3810 3815 3820 Ser Leu Glu Glu Ser Arg Asp Arg Leu Arg Phe Leu Gln Leu His Asn 3825 3830 3835 3840 Trp Leu Asp Asn Arg Ser Arg Ala Val Phe Leu Glu Leu Thr Arg Tyr 3845 3850 3855 Ser Pro Ala Val Gly Leu His Ala Ala Val Thr Leu Arg Leu Glu Phe 3860 3865 3870 Pro Ala Ala Gly Arg Ala Leu Ala Ala Leu Ser Val Arg Pro Phe Ala 3875 3880 3885 Leu Arg Arg Leu Ser Ala Gly Leu Ser Leu Pro Leu Leu Thr Ser Val 3890 3895 3900 Cys Leu Leu Leu Phe Ala Val His Phe Ala Val Ala Glu Ala Arg Thr 3905 3910 3915 3920 Trp His Arg Glu Gly Arg Trp Arg Val Leu Arg Leu Gly Ala Trp Ala 3925 3930 3935 Arg Trp Leu Leu Val Ala Leu Thr Ala Ala Thr Ala Leu Val Arg Leu 3940 3945 3950 Ala Gln Leu Gly Ala Ala Asp Arg Gln Trp Thr Arg Phe Val Arg Gly 3955 3960 3965 Arg Pro Arg Arg Phe Thr Ser Phe Asp Gln Val Ala His Val Ser Ser 3970 3975 3980 Ala Ala Arg Gly Leu Ala Ala Ser Leu Leu Phe Leu Leu Leu Val Lys 3985 3990 3995 4000 Ala Ala Gln His Val Arg Phe Val Arg Gln Trp Ser Val Phe Gly Lys 4005 4010 4015 Thr Leu Cys Arg Ala Leu Pro Glu Leu Leu Gly Val Thr Leu Gly Leu 4020 4025 4030 Val Val Leu Gly Val Ala Tyr Ala Gln Leu Ala Ile Leu Leu Val Ser 4035 4040 4045 Ser Cys Val Asp Ser Leu Trp Ser Val Ala Gln Ala Leu Leu Val Leu 4050 4055 4060 Cys Pro Gly Thr Gly Leu Ser Thr Leu Cys Pro Ala Glu Ser Trp His 4065 4070 4075 4080 Leu Ser Pro Leu Leu Cys Val Gly Leu Trp Ala Leu Arg Leu Trp Gly 4085 4090 4095 Ala Leu Arg Leu Gly Ala Val Ile Leu Arg Trp Arg Tyr His Ala Leu 4100 4105 4110 Arg Gly Glu Leu Tyr Arg Pro Ala Trp Glu Pro Gln Asp Tyr Glu Met 4115 4120 4125 Val Glu Leu Phe Leu Arg Arg Leu Arg Leu Trp Met Gly Leu Ser Lys 4130 4135 4140 Val Lys Glu Phe Arg His Lys Val Arg Phe Glu Gly Met Glu Pro Leu 4145 4150 4155 4160 Pro Ser Arg Ser Ser Arg Gly Ser Lys Val Ser Pro Asp Val Pro Pro 4165 4170 4175 Pro Ser Ala Gly Ser Asp Ala Ser His Pro Ser Thr Ser Ser Ser Gln 4180 4185 4190 Leu Asp Gly Leu Ser Val Ser Leu Gly Arg Leu Gly Thr Arg Cys Glu 4195 4200 4205 Pro Glu Pro Ser Arg Leu Gln Ala Val Phe Glu Ala Leu Leu Thr Gln 4210 4215 4220 Phe Asp Arg Leu Asn Gln Ala Thr Glu Asp Val Tyr Gln Leu Glu Gln 4225 4230 4235 4240 Gln Leu His Ser Leu Gln Gly Arg Arg Ser Ser Arg Ala Pro Ala Gly 4245 4250 4255 Ser Ser Arg Gly Pro Ser Pro Gly Leu Arg Pro Ala Leu Pro Ser Arg 4260 4265 4270 Leu Ala Arg Ala Ser Arg Gly Val Asp Leu Ala Thr Gly Pro Ser Arg 4275 4280 4285 Thr Pro Leu Arg Ala Lys Asn Lys Val His Pro Ser Ser Thr 4290 4295 4300 23 base pairs nucleic acid single unknown cDNA Homo sapiens misc_feature 1..23 /function= ”AH3 F9 primer“ 9 TTTGACAAGC ACATCTGGCT CTC 23 20 base pairs nucleic acid single unknown cDNA Homo sapiens misc_feature 1..20 /function= ”AH3 B7 primer“ 10 TACACCAGGA GGCTCCGCAG 20 21 base pairs nucleic acid single unknown cDNA Homo sapiens misc_feature 1..21 /function= ”3A3 C1 primer“ 11 CGCCGCTTCA CTAGCTTCGA C 21 20 base pairs nucleic acid single unknown cDNA Homo sapiens misc_feature 1..20 /function= ”3A3 C2 primer“ 12 ACGCTCCAGA GGGAGTCCAC 20 20 base pairs nucleic acid single unknown cDNA Homo sapiens misc_feature 1..20 /function= ”AH4F2 primer“ 13 GGGCAAGGGA GGATGACAAG 20 21 base pairs nucleic acid single unknown DNA (genomic) Homo sapiens misc_feature 1..21 /function= ”JH14B3 primer“ 14 GGGTTTATCA GCAGCAAGCG G 21 30 base pairs nucleic acid unknown unknown cDNA Homo sapiens misc_feature 1..30 /function= ”N2765 primer“ 15 GGCGCGGCGG GCGGCATCGT TAGGGCAGCG 30 30 base pairs nucleic acid unknown unknown cDNA Homo sapiens misc_feature 1..30 /function= ”N5496 primer“ 16 GGCGGGCGGC ATCGTTAGGG CAGCGCGCGC 30 30 base pairs nucleic acid unknown unknown cDNA Homo sapiens misc_feature 1..30 /function= ”N5495 primer“ 17 ACCTGCTGCT GAGCGACGCC CGCTCGGGGC 30 54 base pairs nucleic acid unknown unknown genomic DNA Homo sapiens 18 TTTTGGTCAA GGTGAGGGCT GGGCCGGTGG GCGCGGGGCT GGGCGCACAC CCCA 54 554 base pairs nucleic acid unknown unknown cDNA Homo sapiens misc_feature /function= ”1A1H0.6 probe“ 19 AAGCTTGGCA CCATCAAGGG CCAGTTCAAC TTTGTCCACG TGATCGTCAC CCCGCTGGAC 60 TACGAGTGCA ACCTGGTGTC CCTGCAGTGC AGGAAAGACA TGGAGGGCCT TGTGGACACC 120 AGCGTGGCCA AGATCGTGTC TGACCGCAAC CTGCCCTTCG TGGCCCGCCA GATGGCCCTG 180 CACGCAAATA TGGCCTCACA GGTGCATCAT AGCCGCTCCA ACCCCACCGA TATCTACCCC 240 TCCAAGTGGA TTGCCCGGCT CCGCCACATC AAGCGGCTCC GCCAGCGGAT CTGCGAGGAA 300 GCCGCCTACT CCAACCCCAG CCTACCTCTG GTGCACCCTC CGTCCCATAG CAAAGCCCCT 360 GCACAGACTC CAGCCGAGCC CACACCTGGC TATGAGGTGG GCCAGCGGAA GCGCCTCATC 420 TCCTCGGTGG AGGACTTCAC CGAGTTTGTG TGAGGCCGGG GCCCTCCCTC CTGCACTGGC 480 CTTGGACGGT ATTGCCTGTC AGTGAAATAA ATAAAGTCCT GACCCCAGTG CACAGACATA 540 GAGGCACAGA TTGC 554 192 base pairs nucleic acid unknown unknown other nucleic acid Homo sapiens misc_feature /function= ”CW10F probe“ 20 GTCCGCGGTC GCACGTACGC TTCTGGTGTG TGTGAGACGT GCGGGGCTGG GAAGTGTTGG 60 CAGACGGCGA GTACGTCCTC ACTCCTTTTG TTCTTTTGAC CTAAGCTGGC GAGTGGCACT 120 GCTGAGTTCC GCTCAGTGCC CGCCCTGATG TGCGACCCCC GTGCATTCTT GCTGTTAGGT 180 GGTGGCGGTG TG 192 41 base pairs nucleic acid unknown unknown other nucleic acid Homo sapiens misc_feature /function= ”CW10R probe“ 21 AGGCAGGTCT CCCCCACGAC CAGGGGAGAG GCACCCAAGG T 41 31 base pairs nucleic acid unknown unknown other nucleic acid Homo sapiens 22 AGTCAGTAAT TTATATGGTG TTAAAATGTG A 31 6 amino acids amino acid single linear protein Homo sapiens 23 Trp Asp Phe Gly Asp Ser 1 5 

What is claimed is:
 1. An isolated polycystic kidney disease 1 (PKD1) gene having the nucleic acid sequence presented in SEQ ID NO: 7 or a nucleic acid molecule having a sequence complementary to the entire nucleic acid sequence of SEQ ID NO:7.
 2. An isolated polycystic kidney disease 1 (PKD1) gene having the nucleic acid sequence presented in SEQ ID NO: 1 or a nucleic acid molecule having a sequence complementary to the entire nucleic acid sequence of SEQ ID NO:1.
 3. An isolated polycystic kidney disease 1 (PKD1) gene having the nucleic acid sequence presented in SEQ ID NO: 5 or a nucleic acid molecule having a sequence complementary to the entire nucleic acid sequence of SEQ ID NO:5.
 4. An isolated nucleic acid molecule wherein the molecule is an RNA transcript comprising a length of about 14 kB; said RNA transcript having a nucleic acid sequence corresponding to the coding region of the nucleic acid sequence of SEQ ID NO: 7, or a sequence that is complementary to the entire coding region of the nucleic acid sequence of SEQ ID NO:
 7. 5. A recombinant expression vector comprising the isolated nucleic acid of claim 1, 2 or
 3. 6. A host cell comprising the vector of claim
 5. 7. A recombinant expression vector comprising the isolated nucleic acid complementary to the RNA transcript of claim
 4. 8. An isolated nucleic acid isolated from a patient clinically diagnosed with autosomal polycystic kidney disease, which encodes a polycystic kidney disease (PKD1) gene product, and wherein said nucleic acid comprises a mutant PKD1 gene comprising a deletion of at least 18 contiguous nucleotides as compared to the PKD1 sequence presented in SEQ ID NO:
 7. 9. A recombinant expression vector containing a polynucleotide consisting of SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO:
 7. 10. A host cell comprising the vector of claim
 9. 